Immunizing compositions and methods of use

ABSTRACT

The present invention provides compositions including siderophore receptor polypeptides and porins from gram negative microbes, and preferably, lipopolysaccarhide at a concentration of no greater than about 10.0 endotoxin units per milliliter. The present invention also provides methods of making and methods of using such compositions.

CONTINUING APPLICATION DATA

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/259,504, filed Jan. 3, 2001, and U.S.Provisional Application Ser. No. 60/262,896, filed Jan. 19, 2001 whichis incorporated by reference herein.

BACKGROUND

[0002] The economic impact of infectious diseases in food animalproduction is well appreciated. Infectious diseases reduce profits,increase production costs, and endanger the overall wholesomeness of thefood products, as well as effect the performance, health and welfare ofthe animal. This disease status can reduce the yield and quality of milkresulting in great economic loss to the dairymen. In some cases,infectious microbial diseases can cause morbidity and mortality ofnewborn, young (e.g., replacement stock) or adult animals.

[0003] The agricultural industry presently relies on antibiotic therapyand vaccines to decrease losses caused by clinical and subclinicalinfectious diseases, including gastrointestinal disease, respiratorydisease, and systemic disease. However, for some conditions, antibioticsare ineffective, may prolong the condition, or induce a carrier state.Vaccines have often proven to be an effective means of controllinginfectious diseases, but, concerns relating to adverse effects or lackof protection against multiple microbes have been a major drawback tocurrent vaccines. For instance, vaccines are available that contain oneor more immunogens against an individual genus, species, or strain ofmicrobe; however, few, if any, provide cross-protection or stimulatebroad-based immunity against multiple strains, species or genera ofmicrobe.

[0004] Vaccines containing molecules obtained from gram negativemicrobes typically include contaminating levels of lipopolysaccharide(LPS), a component of the outer membrane of most gram negative microbes.The presence of LPS in an injectable product can result in aninflammatory response at the site of injection that can result inswelling, tenderness and often the formation of a granuloma at the siteof injection. In rare cases, it can result in anaphylactic shock anddeath. This non-specific inflammatory response in a production animalcan result in significant economic losses due to increasing thelikelihood of disease by increasing the level of stress of the animal,and negatively effecting performance characteristics of the animal. Inaddition, the formation of a granuloma at the injection site can resultin significant economic losses due to blemishes and scarring of thecarcass which are often trimmed during processing resulting in the lossof product and down grading of the carcass. While methods for removal ofLPS from compositions exist, this is often not feasible for use withvaccines that include whole cells. Moreover, due to the high costs ofremoving LPS from solutions, it is typically not economically practicalto remove LPS from vaccines for use in non-human animals.

SUMMARY OF THE INVENTION

[0005] The presence of LPS in animal vaccines has a significant economicimpact. However, the refusal of farmers to pay high fees for vaccineshas prevented the use of available, but costly, methods for LPS removal.Accordingly, there is a long standing but unresolved need for methodsfor economically producing compositions containing molecules from gramnegative microbes that contain low amounts of contaminating LPS. Thepresent invention represents an advance in the art of economicallyisolating polypeptides from gram negative microbes with low levels ofcontaminating LPS. Accordingly, the present invention provides methodsfor isolating outer membrane polypeptides. The method includes providinga gram negative microbe, disrupting the gram negative microbe in abuffer, solubilizing the disrupted gram negative microbe, and isolatingmolecules of the gram negative microbe, wherein the isolated moleculesinclude outer membrane polypeptides including at least two siderophorereceptor polypeptides (SRPs) and at least two porins, and LPS at aconcentration of no greater than about 10.0 endotoxin units permilliliter (EU/ml). During disrupting, the gram negative microbe may bepresent in the buffer at a concentration of between about 720 grams ofmicrobe per 1,000 milliliters of buffer and about 1,080 grams of microbeper 1,000 milliliters of buffer. Solubilization of the gram negativemicrobe may occur for greater than about 24 hours. Solubilization of thegram negative microbe may occur in a solution including sarcosine, wherethe ratio of the sarcosine to gram weight of disrupted gram negativemicrobe is between about 0.8 gram sarcosine per about 4.5 grams ofdisrupted gram negative microbe and about 1.2 grams sarcosine per about4.5 grams of disrupted gram negative microbe.

[0006] The present invention is also directed to a composition includingat least two SRPs isolated from a gram negative microbe, at least twoporins isolated from the gram negative microbe, and LPS at aconcentration of no greater than about 10.0 EU/ml. The composition mayfurther include a pharmaceutically acceptable carrier. The gram negativemicrobe may be an enteropathogen, preferably, a member of the familyEnterobacteriaceae, more preferably, a member of the tribe Escherichieaeor Salmonelleae, most preferably, Salmonella spp. or Escherichia coli.The at least two SRPs may have molecular weights of between about 60 kDaand about 100 kDa, and the at least two porins may have molecularweights of between about 30 kDa and about 43 kDa.

[0007] The present invention also represents an advance in the art ofstimulating immunity to multiple strains, species, or genera of microbe.Accordingly, the present invention also provides a method for inducingthe production of antibody in an animal. The method includesadministering to an animal an effective amount of a composition of thepresent invention further including a pharmaceutically acceptablecarrier, where the composition induces in the animal antibody thatspecifically binds at least one SRPs or at least one porin. The gramnegative microbe may be an enteropathogen, preferably, a member of thefamily Enterobacteriaceae, more preferably, a member of the tribeEscherichieae or Salmonelleae, most preferably, Salmonella spp. orEscherichia coli. The animal may be an avian, a bovine, a caprine, aporcine, or an ovine. When the animal is a bovine, the bovine mayexhibit a phenotype of, for instance, decreased somatic cell count,increased milk production, decreased fecal shedding, or increasedweight.

[0008] The present invention is further directed to a method forinducing the production of antibody in an animal, where the methodincludes administering to an animal an effective amount of a compositionthat includes at least four SRPs isolated from a gram positive microbeand a pharmaceutically acceptable carrier, where the composition inducesin the animal antibody to the SRP. The gram positive microbe may be amember of the family Micrococcaceae, for instance, Staphylococcusaureus. The SRPs may have molecular weights of between about 60 kDa andabout 100 kDa.

[0009] Also provided by the present invention are methods for treatingconditions in an animal, including, for instance, a high somatic cellcount, fecal shedding of a microbe in an animal's intestinal tract, lowmilk production, mastitis in a milk producing animal, and metritis in ananimal. The methods include administering to an animal having or at riskof having the condition an effective amount of a composition of thepresent invention, where the composition further includes apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1. Comparison of Salmonella isolation and serologicalresponse to vaccination in lactating cows. Percent positive isolation,percent of vaccinated lactating cows shedding Salmonella bredeney;antibody response (O.D.), optical density at 405 nm of antibody responseas measured by ELISA. The bars correspond to the y-axis on the left(Percent Positive Isolation) and the open diamonds correspond to they-axis on the right (Antibody Response (O.D.)).

[0011]FIG. 2. The Dairy Herd Improvement Association (DHIA) somatic cellcount on individual cows before and after the first vaccination. Averagecell count X 1,000, average somatic cell count times 1,000; Cowssampled, identification of each of the 51 cows; Pre-Vac, average cellcount X 1,000 of each cow before vaccination; Post-Vac, average cellcount X 1,000 of each cow before vaccination.

[0012]FIG. 3. Cumulative pounds of milk produced before and after thethird vaccination. Bulk Tank Average (lbs), pounds of milk produced byall cows in lactation. The shaded areas “Before vaccination” and “1.2%After vaccination” represent the difference in percent in milkproduction before and after the third vaccination.

[0013]FIG. 4. The cumulative rolling herd average showing pounds of milkproduced before and after vaccination in lactating cows. Pounds of milk,pounds of milk produced by all cows in lactation and averaged over theperiod of a month. Shaded area represents the rise in milk productionduring vaccination.

[0014]FIG. 5. The average monthly cost in antibiotic usage before andafter vaccination. 51% reduction refers to the reduction of the costs ofantibiotics after vaccination.

[0015]FIG. 6. The average weekly milk production between vaccinated andnon-vaccinated cows in first lactation. Weekly Milk Production/Group,weekly production of milk (in pounds) for the control group and thevaccinated cows.

[0016]FIG. 7. The average weekly milk production between vaccinated andnon-vaccinated fresh cows. Weekly Milk Production/Group, weeklyproduction of milk (in pounds) for the control group and the vaccinatedcows.

[0017]FIG. 8. The monthly average somatic cell count (DHIA) betweenvaccinated and non-vaccinated cows in first lactation.

[0018]FIG. 9. The monthly average somatic cell count (DHIA) betweenvaccinated and non-vaccinated fresh cows.

[0019]FIG. 10. The serological response of vaccinated steers compared tonon-vaccinated controls. Mean optical density (O.D.), 405 nm;P3-Vaccinated and P5-Vaccinated, vaccinated steers in pens 3 and 5,respectively; P4-Control and P6-Control, non-vaccinated steers in pens 4and 6, respectively; Sampling time, weeks after first vaccination.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0020] Compositions

[0021] One aspect of the present invention provides compositionsincluding siderophore receptor polypeptides (SRPs) and porins obtainedfrom a microbe. Unless otherwise specified, the term “microbe” includesboth gram negative microbes and gram positive microbes. As used herein,“polypeptide” refers to a polymer of amino acids linked by peptide bondsand does not refer to a specific length of a polymer of amino acids.Thus, for example, the terms peptide, oligopeptide, protein, and enzymeare included within the definition of polypeptide. This term alsoincludes post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations, and the like. Apolypeptide can be produced using recombinant techniques, or chemicallyor enzymatically synthesized. Preferably, the polypeptides of thecompositions of the present invention are isolated. An “isolated”polypeptide means a polypeptide that has been either removed from itsnatural environment, produced using recombinant techniques, orchemically or enzymatically synthesized. Unless otherwise specified,“a,” “an,” “the,” and “at least one” are used interchangeably and meanone or more than one.

[0022] Gram negative microbes suitable for use in obtaining SRPs arethose capable of producing SRPs when incubated under low ironconditions. Low iron conditions are described herein. Such gram negativemicrobes include enteropathogens, preferably, members of the familyEnterobacteriaceae, more preferably, members of the familyEnterobacteriaceae that are members of the tribe Escherichieae orSalmonelleae, even more preferably, E. coli or Salmonella spp. Examplesof preferred enteropathogens include members of the familyEnterobacteriaceae, members of the family Vibrionaceae (including, forinstance, Vibrio cholerae), and Campylobacter spp. (including, forinstance, C. jejuni). Examples of preferred members of the familyEnterobacteriaceae include, for instance, E. coli, Shigella spp.,Salmonella spp., Proteus spp., Klebsiella spp. (for instance, Klebsiellapneumoniae), Serratia spp., and Yersinia spp. Preferred examples ofSalmonella spp. include Salmonella enterica serovars, Bredeney, Dublin,Agona, Blockley, Enteriditis, Typhimurium, Hadar, Heidelberg,Montevideo, Muenster, Newport senftenberg, Salmonella cholerasuis, andS. typhi. Salmonella enterica serovars Bredeney, Dublin and Typhimuriumare referred to herein as Salmonella bredeney, S. dublin, and S.typhimurium, respectively. Preferred examples of strains of E. coliinclude, for example, E. coli serotypes O1a, O2a, O78, and O157,different O:H serotypes including 0104, 0111, 026, 0113, 091, andhemolytic strains of enterotoxigenic E. coli such as K88⁺, F4⁺, F18ab⁺,and F18ac⁺. As used herein, the term “strain” refers to members of aspecies of microbe where the members have different genotypes and/orphenotypes. Other gram negative microbes include members of the familyPasteurellaceae, preferably Pasturella spp., more preferably, Pasturellamultocida and Pasteurella haemolytica, and members of the familyPseudomonadaceae, preferably Pseudomonasspp., most preferably,Pseudomonas aeruginosa, Yet other gram negative microbes includeActinobacillus spp., Haemophilus spp., Myxcobacteria spp.,Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacterspp., Flavobacterium spp., Aeromonas spp., among other gram-negativebacteria.

[0023] Gram positive microbes from which polypeptides may be obtainedinclude members of the family Micrococcaceae, preferably, Staphylococcusspp., more preferably, Staphylococcus aureus. Other gram positivemicrobes include members of the family Deinococcaceae, preferably,Streptococcus agalactiae, Streptococcus uberis, Streptococcus bovis,Streptococcus equi, Streptococcus zooepidemicus, or Streptococcusdysgalatiae. Other gram positive microbes from which polypeptides can beisolated include Bacillus spp., Clostridium spp., Corynebacterium spp.,Erysipelothrix spp., Listeria spp., and Mycobacterium spp.,Erysipelothrix spp., and Clostridium spp.

[0024] These microbes are commercially available from a depository suchas American Type Culture Collection (ATCC). In addition, such microbesare readily obtainable by isolation techniques known and used in theart. The microbes may be derived from an infected animal as a fieldisolate, and screened for production of SRPs, and introduced directlyinto low iron conditions, or stored for future use, for example, in afrozen repository at about −20° C. to about −95° C., preferably about40° C. to about −50° C., in bacteriological media containing 20%glycerol, and other like media.

[0025] The present invention provides compositions including at leasttwo, preferably, at least three, siderophore receptor polypeptides(SRPs). SRPs of gram negative microbes are polypeptides present in theouter membrane of gram negative microbes, and SRPs of gram positivemicrobes are polypeptides present in the membrane of gram positivemicrobes. In some aspects of the invention, SRPs are expressed by amicrobe at high levels when the microbe is exposed to low ironconditions, and expressed at a substantially lower level when themicrobe is exposed to high iron conditions. Preferably, SRPs areexpressed by a microbe when the microbe is exposed to low ironconditions, and not expressed at detectable levels when the microbe isexposed to high iron conditions. Low iron conditions and high ironconditions are described in greater detail herein. Without intending tobe limited by theory, it is believed that the SRPs of the presentcompositions are receptors of iron-binding siderophores. Examples ofsiderophore receptors expressed by gram negative microbes include, forinstance, receptors for the uptake of aerobactin, enterobactin, ferriccitrate, ferrichrome, rhodotorulic, and coprogen, as well as receptorsfor the transferrins (for instance the serotransferrins,lactotransferrin, and ovotransferrin), and other binding proteins, (see,for instance, Emery et al., U.S. Pat. No. 5,830,479, and Crichton,Microbial Iron Uptake and Intracellular Release. In: InorganicBiochemistry of Iron Metabolism, Burgess, (ed)., Ellis Horwood Limited,Chichester, England, 59-76 (1991)).

[0026] Preferably, SRPs of the compositions of the present inventionhave immunogenic activity. “Immunogenic activity” refers to the abilityof a polypeptide to elicit an immunological response in an animal. Animmunological response to a polypeptide is the development in an animalof a cellular and/or antibody-mediated immune response to thepolypeptide. Usually, an immunological response includes but is notlimited to one or more of the following effects: the production ofantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells, directed to an epitope or epitopes of thepolypeptide. “Epitope” refers to the site on an antigen to whichspecific B cells and/or T cells respond so that antibody is produced.

[0027] It is known to the art that receptors of siderophores typicallyinclude epitopes that are conserved in the SRPs of different species anddifferent genera of microbes (see, for instance, Emery et al. (U.S. Pat.No. 5,830,479) and Example 8). For instance, antibodies produced againstan aerobactin receptor protein of one species, strain or genus of thefamily Enterobacteriaceae (for instance, E. coli, Salmonella spp., andKlebsiella spp.) have been found to cross-react with other microbeswithin the family. Species of Pseudomonas of the family Pseudomonadaceaealso express siderophore receptor proteins that can be isolated asdescribed herein and produce antibodies that cross-react with thereceptor proteins of E. coli, Salmonella spp., and Klebsiella spp.,among other members of the family Enterobacteriaceae. Moreover,antibodies produced against SRPs of Salmonella and against SRPs of E.coli have been found to cross react with the gram positive microbeStaphylococcus aureus (see Example 11).

[0028] A composition of the present invention may contain at least two,preferably, at least three, SRPs isolated from one or more genera or oneor more species of microbe. In some aspects of the present invention,preferably the SRPs of a composition are derived from multiple speciesof the same genus of microbe, or from multiple strains of the samespecies of microbe. The present invention also includes compositionsincluding SRPs isolated from at least one gram negative microbe and atleast one gram positive microbe. Preferably, the molecular weights ofSRPs, as determined by separation of the SRPs using an about 12% sodiumdodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) gel under reducingand denaturing conditions, are between about 60 kDa (kiloDaltons) andabout 100 kDa, more preferably, between about 65 kDa and about 95 kDa.

[0029] Typically, different species of Salmonella each produce threeSRPs. Without intending to be limited by theory, it is believed that thethree SRPs produced by Salmonella spp. are receptors for thesiderophores enterochelin, aerobactin, and ferrichrome. Preferably, SRPsobtained from S. dublin and S. typhimurium are combined. Preferably, themolecular weights of SRPs isolated from Salmonella, as determined byseparation of the SRPs using an about 12% SDS-PAGE gel under reducingand denaturing conditions, are between about 60 kDa and about 100 kDa,more preferably, between about 65 kDa and about 95 kDa. More preferably,the molecular weights of SRPs isolated from Salmonella are as follows:between about 87 kDa and about 91 kDa, preferably about 89 kDa; betweenabout 82 kDa and about 86 kDa, preferably about 84 kDa; and betweenabout 69 kDa and about 75 kDa, preferably about 72 kDa.

[0030]E. coli have been found to produce 2, 3, 4, or 6 SRPs, dependingon the serotype. Preferably, a composition that includes SRPs from E.coli includes, in increasing preference, at least two, at least three,at least four, or at least six SRPs isolated from E. coli. SRPs isolatedfrom different E. coli strains can be combined. Preferably, themolecular weights of SRPs isolated from an E. coli, as determined byseparation of the SRPs using an about 12% SDS-PAGE gel under reducingand denaturing conditions, are between about 60 kDa and about 100 kDa,more preferably, between about 65 kDa and about 95 kDa. More preferably,in a composition including SRPs isolated from an E. coli, the SRPs havemolecular weights selected from between about 91 kDa and about 93 kDa,preferably about 92 kDa; between about 88 kDa and about 90 kDa,preferably about 89 kDa; between about 82 kDa and about 86 kDa,preferably about 84 kDa; between about 76 kDa and about 80 kDa,preferably about 78 kDa; between about 73 kDa and about 75 kDa,preferably about 74 kDa; and between about 71 kDa and about 73 kDa,preferably about 72 kDa. A preferred composition that includes SRPsisolated from E. coli is isolated from the E. coli deposited with theAmerican Type Culture Collection, 10801 University Blvd., Manassas, Va.,20110-2209, USA, on Dec. 29, 1994, and designated ATCC #55652. Thedeposit was made under the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure.

[0031] Field isolates of the gram positive microbe Staphylococcus aureushas been found to produce at least about 4 SRPs. Preferably, when thecomposition includes SRPs from S. aureus, the SRPs are isolated from atleast one species of S. aureus, more preferably, from one species of S.aureus. Preferably, the S. aureus is isolated from an avian animalsuffering from a disease caused by S. aureus. Preferably, the molecularweights of four of the SRPs isolated from an S. aureus, as determined byseparation of the SRPs using an about 10% SDS-PAGE gel under reducingand denaturing conditions, are between about 60 kDa and about 100 kDa,more preferably, between about 65 kDa and about 95 kDa. More preferably,the molecular weights of SRPs isolated from S. aureus are as follows:between about 88 kDa and about 92 kDa, preferably about 90 kDa; betweenabout 82 kDa and about 86 kDa, preferably about 84 kDa; between about 70kDa and about 74 kDa, preferably about 72 kDa; and between about 64 kDaand about 68 kDa, preferably about 66 kDa. Preferably, the molecularweights of the other three SRPs isolated from S. aureus are betweenabout 35 kDa and about 37 kDa, preferably about 36 kDa; between about 30kDa and about 34 kDa, preferably about 32 kDa; and between about 20 kDaand about 24 kDa, preferably about 22 kDa. Preferably, an S. aureus fromwhich the SRPs are isolated is obtained from a bird, for instance achicken or a turkey, displaying symptoms of a disease caused by the S.aureus, for instance, septicemia.

[0032] Preferably, SRPs of the present compositions can be identifiedusing antibodies that specifically bind SRPs. As used herein, anantibody that can “specifically bind” a polypeptide is an antibody thatinteracts with the epitope of the antigen that induced the synthesis ofthe antibody, or interacts with a structurally related epitope. Suchantibodies can be made using the E. coli strain having the designationATCC #55652. Typically, ATCC #55652 is grown under low iron conditions,and SRPs are isolated from the strain as described in Example 1, or asdescribed by, for example, Emery et al. (U.S. Pat. No. 5,830,479).Antibody is then made that specifically binds the SRPs using laboratorymethods for producing polyclonal and monoclonal antibodies. Suchlaboratory methods are routine and known in the art (see, for instance,Harlow E. et al. Antibodies: A laboratory manual Cold Spring HarborLaboratory Press, Cold Spring Harbor (1988) and Ausubel, R. M., ed.Current Protocols in Molecular Biology (1994)). Methods for determiningwhether SRPs of the present compositions are specifically bound byantibodies made using SRPs isolated from ATCC #55652 are routine andknown to the art, and include, for instance, western immunoblot andenzyme linked immunosorbant assay.

[0033] The compositions of the present invention also include at leasttwo porin polypeptides. Porin polypeptides are transmembraneous poreforming-proteins of the outer membrane of gram negative microbes. Gramnegative bacteria have a cell wall with a thin peptidoglycan membranelayer in which small hydrophilic compounds can diffuse through the outermembrane by the porin pathway. Gram positive microbes have a thickpeptidoglycan layer, which is porous and does not form a permeabilitybarrier on the surface. It has been widely accepted that gram positivebacteria do not possess well-defined pore-forming proteins as comparedto gram-negative bacteria. Nevertheless, recent evidence has shownidentification of channel-forming activity in some members of the familyCorynebacteriaceae. Unlike SRPs, the expression of porins does notchange in response to the level of iron present in the medium in which amicrobe is grown, and porin expression is typically constitutive.Without intending to be limited by theory, it is believed that theporins of the present compositions are polypeptides that produce poresor channels allowing passage of molecules across the outer membrane ofgram negative microbes (see, for instance, Nikaido and Vaara, OuterMembrane, In: Escherichia coli and Salmonella typhimurium, Cellular andMolecular Biology, Neidhardt et al., (eds.) American Society forMicrobiology, Washington, D.C., pp. 7-22 (1987)) and the membrane ofgram positive microbes. For instance, it is believed that the porinsproduced by gram negative microbes may include OmpA, OmpC, OmpD, OmpF,or PhoE. The porins are relatively conserved between gram negativebacteria, and play a role in iron binding. For example, OmpF and OmpCwill bind lactoferrin (Erdei et al., Infec. Immun., 62, 1236-1240(1994)), while OmpA will bind ferrichrome (Coulton et al., J. Gen.Microbiol., 110, 211-220 (1979)). Antibodies early in infectionparticularly of the IgM class have been found to cross-react with porinsof E. coli, Salmonella, Pasteurella, Pseudomonas and Klebsiella, andwill bind lactoferrin and/or ferrichrome, precluding the availability ofan iron source for microbial growth. Without intending to be limited bytheory, antibodies to these polypeptides will also bind to the porins onthe surface to enhance opsonization and/or complement-mediated bacteriallysis.

[0034] A composition of the present invention may contain at least twoporins isolated from one or more genera or one or more species ofmicrobe. In some aspects of the present invention, preferably the porinsof a composition are derived from multiple species of microbes of thesame genus of microbe, or from multiple strains of the same species ofmicrobe. In some aspects of the present invention, preferably the porinsof a composition are derived from the same microbe from which the SRPsof the composition were isolated.

[0035] Preferably, porins of the compositions of the present inventionhave immunogenic activity. Without intending to be limiting, porins ofthe present composition act as an adjuvant to enhance the immuneresponse of an animal to porins and SRPs present in a composition of thepresent invention when administered to an animal as described herein.

[0036] Preferably, the molecular weights of porins of the compositionsof the present invention, as determined by separation of the porinsusing an about 12% SDS-PAGE gel under reducing and denaturingconditions, are between about 30 kDa and about 43 kDa, more preferably,between about 33 kDa and about 40 kDa. Preferably, the porins areobtained from a gram negative microbe. Typically, different species ofSalmonella each produce at least two porins. Preferably, when thecomposition includes porins from a Salmonella, the porins are isolatedfrom one species of Salmonella. Preferably, the molecular weights ofporins isolated from Salmonella spp. are between about 37 kDa to about40 kDa, more preferably, between about 38 kDa and about 39 kDa.Typically, E. coli produces at least two porins. Preferably, themolecular weights of porins isolated from E. coli are between about 33kDa to about 39 kDa, more preferably, between about 34 kDa and about 38kDa.

[0037] Preferably, porins of the present compositions can be identifiedusing antibodies that specifically bind porins. Such antibodies can bemade using the E. coli strain having the designation ATCC #55652.Typically, ATCC #55652 is grown under low iron conditions, and porinsare isolated from the strain as described in Example 1, or as describedby Emery et al. (U.S. Pat. No. 5,830,479). Antibody is then made thatspecifically binds the porins. Laboratory methods for producingpolyclonal and monoclonal antibodies are routine and known in the art(see, for instance, Harlow E. et al. Antibodies: A laboratory manualCold Spring Harbor Laboratory Press, Cold Spring Harbor (1988) andAusubel, R. M., ed. Current Protocols in Molecular Biology (1994)).Methods for determining whether porins of the present compositions arespecifically bound by antibodies made using porins isolated from ATCC#55652 are routine and known to the art, and include, for instance,western immunoblot and enzyme linked immunosorbant assay.

[0038] Preferably, the compositions of the present invention include lowconcentrations, more preferably, undetectable concentrations, oflipopolysaccharide (LPS). LPS is a component of the outer membrane ofmost gram negative microbes (see, for instance, Nikaido and Vaara, OuterMembrane, In: Escherichia coli and Salmonella typhimurium, Cellular andMolecular Biology, Neidhardt et al., (eds.) American Society forMicrobiology, Washington, D.C., pp. 7-22 (1987), and typically includespolysaccharides (O-specific chain, the outer and inner core) and thelipid A region. The lipid A component of LPS is the most biologicallyactive component of the LPS structure and together induce a widespectrum of pathophysiological effects in mammals. The most dramaticeffects are fever, disseminated intravascular coagulation, complementactivation, hypotensive shock, and death. LPS plays a major role in theactivation of various cell types, particularly those of lymphoid origin.This activation results in the production of an impressive array ofendogenous mediators that, in turn, activate the complement system,impair mitochondrial function, activate lysosomal activity, stimulateprotaglandin activity, and cause macrophage cytotoxicity and tumoricidalactivity. This non-specific immunostimulatory activity of LPS canenhance the formation of a granuloma at the site of administration ofcompositions that include LPS. Such reactions can result in undue stresson the animal by which the animal may back off feed or water for aperiod of time, and exasperate infectious conditions in the animal. Inaddition, the formation of a granuloma at the site of injection canincrease the likelihood of possible down grading of the carcass due toscaring or blemishes of the tissue at the injection site (see, forinstance, Rae, Injection Site Reactions, available atwww.animal.ufl.edu/short94/rae.htm).

[0039] The concentration of LPS can be determined using routine methodsknown to the art. Such methods typically include measurement of dyebinding by LPS (see, for instance, Keler and Nowotny, Analyt. Biochem.,156, 189 (1986)) or the use of a Limulus amebocyte lysate (LAL) test(see, for instance, Endotoxins and Their Detection With the LimulusAmebocyte Lystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York,N.Y. (1982)). There are four basic commercially available methods thatare typically used with an LAL test: the gel-clot test; theturbidimetric (spectrophotometric) test; the colorimetric test; and thechromogenic test. An example of a gel-clot assay is available under thetradename E-TOXATE (Sigma Chemical Co., St. Louis, Mo.; see SigmaTechnical Bulletin No. 210). Typically, assay conditions includecontacting the composition with a preparation containing a lysate of thecirculating amebocytes of the horseshoe crab, Limulus polyphemus. Whenexposed to LPS, the lysate increases in opacity as well as viscosity andmay gel. About 0.1 milliliter of the composition is added to lysate.Typically, the pH of the composition is between 6 and 8, preferably,between 6.8 and 7.5. The mixture of composition and lysate is incubatedfor about 1 hour undisturbed at about 37° C. After incubation, themixture is observed to determine if there was gelation of the mixture.Gelation indicates the presence of endotoxin. To determine the amount ofendotoxin present in the composition, dilutions of a standardizedsolution of endotoxin are made and tested at the same time that thecomposition is tested. Standardized solutions of endotoxin arecommercially available from, for instance, Sigma Chemical (Catalog No.210-SE) and U.S. Pharmacopeia (Rockville, Md., Catalog No. 235503). Inincreasing order of preference, a composition of the present inventionhas no greater than about 10.0 endotoxin units per milliliter (EU/ml),no greater than about 5.0 EU/ml, no greater than about 1.0 EU/ml, nogreater than about 0.5 EU/ml, no greater than about 0.2 EU/ml, nogreater than about 0.1 EU/ml, most preferably, no greater than about0.05 EU/ml. An endotoxin unit (EU) is defined in comparison to thecurrent FDA Endotoxin Reference Standard Lot EC-5. One vial of lot EC-5contains 10,000 EU. In general, about 1 nanogram (ng) of pure LPS isequal to between about 5 and about 10 endotoxin units.

[0040] The compositions of the present invention optionally furtherinclude a pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable” refers to a diluent, carrier, excipient, salt, etc, that iscompatible with the other ingredients of the composition, and notdeleterious to the recipient thereof. Typically, the compositionincludes a pharmaceutically acceptable carrier when the composition isused as described below in “Methods of Use.” The compositions of thepresent invention may be formulated in pharmaceutical preparations in avariety of forms adapted to the chosen route of administration,preferably, routes suitable for stimulating an immune response to anantigen. Thus, a composition of the present invention can beadministered via known routes including, for example, oral; parentalincluding intradermal, subcutaneous, intramuscular, intravenous,intraperitoneal, etc., and topically, such as, intranasal,intrapulmonary, intramammary, intravaginal, intrauterine, etc. It isforeseen that a composition can be administered to a mucosal surface,such as by administration to the nasal or respiratory mucosa (e.g. sprayor aerosol), to stimulate mucosal immunity, such as production ofsecretory IgA antibodies, throughout the animal's body.

[0041] A composition of the present invention can also be administeredvia a sustained or delayed release implant. Suitable implants are known.Some examples of implants suitable for use according to the inventionare disclosed in Emery and Straub (WO 01/37810). Implants can beproduced at sizes small enough to be administered by aerosol or spray.Implants also include nanospheres and microspheres.

[0042] A composition of the present invention is administered in anamount sufficient to provide an immunological response to SRPs and/orporins present in the composition, and/or increase performancecharacteristics. Performance characteristics are described in greaterdetail herein. The amount of the polypeptide present in a composition ofthe present invention can vary. For instance, the dosage of polypeptidecan be between about 0.01 micrograms (μg) and about 300 milligrams (mg),typically between about 0.1 mg and about 10 mg. For an injectablecomposition (e.g. subcutaneous, intramuscular, etc.) the polypeptide ispreferably present in the composition in an amount such that the totalvolume of the composition administered is about 0.5 ml to 5.0 ml,typically about 1.0-2.0 ml. The amount administered will vary dependingon various factors including, but not limited to, the specificpolypeptides chosen, the weight, physical condition and age of theanimal, and the route of administration. Thus, the absolute weight ofthe polypeptide included in a given unit dosage form can vary widely,and depends upon factors such as the species, age, weight and physicalcondition of the animal, as well as the method of administration. Suchfactors can be determined by one of skill in the art. Other examples ofdosages suitable for the invention are disclosed in Emery et al. (U.S.Pat. No. 6,027,736).

[0043] The formulations may be conveniently presented in unit dosageform and may be prepared by methods well known in the art of pharmacy.All methods of preparing a composition including a pharmaceuticallyacceptable carrier include the step of bringing the active compound(e.g., SRPs and/or porins as described herein) into association with acarrier that constitutes one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing theactive compound into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product intothe desired formulations.

[0044] A composition including a pharmaceutically acceptable carrier canalso include an adjuvant. An “adjuvant” refers to an agent that can actin a nonspecific manner to enhance an immune response to a particularantigen, thus potentially reducing the quantity of antigen necessary inany given immunizing composition, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Adjuvants may include for example, IL-1, IL-2,emulsifiers, muramyl dipeptides, dimethyldiocradecylammonium bromide(DDA), pyridine, aluminum hydroxide, oils, saponins, alpha-tocopherol,polysaccharides, emulsified paraffins (available from under thetradename EMULSIGEN from MVP Laboratories, Ralston, Nebr.), ISA-70, RIBIand other substances known in the art.

[0045] In another embodiment, an composition of the invention includinga pharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha,IFN-gamma, and other cytokines that effect immune cells. An immunizingcomposition can also include an antibiotic, preservative, anti-oxidant,chelating agent, etc. Such components are known in the art.

[0046] Another aspect of the present invention provides improved methodsfor obtaining SRPs from gram negative microbes and improved methods forobtaining porin polypeptides from gram negative microbes. The methodsinclude providing a gram negative microbe, disrupting the microbe,solubilizing the microbe, and isolating the polypeptides.

[0047] A gram negative microbe to be provided in the method is incubatedunder conditions that promote the expression of SRPs. Typically, suchconditions are low iron conditions. As used herein, the phrase “low ironconditions” refers to an environment, typically bacteriological media,that contains amounts of free iron that cause a microbe to express SRPs.As used herein, the phrase “high iron conditions” refers to anenvironment that contains amounts of free iron that cause a microbe tonot express SRPs. Preferably, low iron conditions are the result of theaddition of an iron chelating compound to media, and high ironconditions are present when a chelator is not present in the media.Examples of iron chelators include 2,2′-dipyridyl (also referred to inthe art as α,α′-bipyridyl), 8-hydroxyquinoline,ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxaminemethanesulphonate (desferol), transferrin, lactoferrin, ovotransferrin,biological siderophores, such as, the catecholates and hydroxamates, andcitrate. Preferably, 2,2′-dipyridyl is used. Typically, 2,2′-dipyridylis added to the media at a concentration of about 25micrograms/milliliter (μg/ml), more preferably, at about 50 μg/ml, mostpreferably, at about 1001 g/ml. The media used to incubate the microbeis not critical, and varies depending on the microbe. For instance, whenthe microbe is Salmonella spp. or E. coli, tryptic soy broth or brainheart infusion may be used. The volume of media used to incubate themicrobe can vary. When a microbe is being evaluated for the ability toproduce SRPs and porins, the microbe can be grown in a suitable volume,for instance, 10 milliliters to 1 liter of medium. When a microbe isbeing grown to obtain SRPs and porins for use in, for instance,administration to animals, the microbe may be grown in a fermentor toallow the isolation of larger amounts of polypeptides. Methods forgrowing microbes in a fermentor are routine and known to the art. Theconditions used for growing a microbe preferably include an ironchelator, preferably 2,2′-dipyridyl, a pH of between about 6.5 and about7.5, preferably between about 6.9 and 7.1, and a temperature of about37° C. Optionally, when a fermentor is used, dissolved oxygen ismaintained at between about 20% and about 40%, preferably, about 30%,but may vary depending on the metabolic requirements of the organism.

[0048] After growth, the gram negative microbe that is to be provided inthe method is harvested. Harvesting includes concentrating the microbeinto a smaller volume and suspending in a media different than thegrowth media. Methods for concentrating a microbe are routine and knownto the art, and include, for example, centrifugation. Typically, theconcentrated microbe is suspended in decreasing amounts of buffer.Preferably, the final buffer includes a metal chelator, preferably,ethylenediaminetetraacetic acid (EDTA), which also aids in the releaseof lipopolysaccharide from the cell wall. Preferably, the final bufferalso minimizes proteolytic degradation. This can be accomplished byhaving the final buffer at a pH of greater than about 8.0, preferably,at least about 8.5, and/or including one or more proteinase inhibitors(e.g., phenylmethanesulfonyl fluoride). Optionally and preferably, theconcentrated microbe is frozen at −20° C. or below until disrupted.

[0049] The gram negative microbe may be disrupted using chemical,physical, or mechanical methods routine and known to the art, including,for example, french press, sonication, or homoginization. Preferably,homoginization is used. As used herein, “disruption” refers to thebreaking up of the cell. Disruption of a microbe can be measured bymethods that are routine and known to the art, including, for instance,changes in optical density. Typically, a microbe is subjected todisruption until the optical density does not change after furtherdisruption. For instance, if percent transmittance is measured, themicrobe is disrupted until the percent transmittance does not increaseafter further disruption. Preferably, the microbe is present in a bufferthat minimizes proteolytic degradation. Preferably, the microbe ispresent in the buffer at a concentration of between about 720 grams ofmicrobe per 1,000 milliliters of buffer and about 1,080 grams of microbeper 1,000 milliliters of buffer, more preferably, between about 810grams of microbe per 1,000 milliliters of buffer to about 990 grams ofmicrobe per 1,000 milliliters of buffer, most preferably, about 900grams microbe per 1,000 milliliters of buffer. The temperature duringdisruption is typically kept low, preferably at about 4° C., to furtherminimize proteolytic degradation.

[0050] The disrupted microbe is solubilized in a detergent, forinstance, an anionic, zwitterionic, nonionic, or cationic detergent.Preferably, the detergent is sarcosine, more preferably, sodium lauroylsarcosinate. As used herein, the term “solubilize” refers to dissolvingcellular materials (e.g., polypeptides, nucleic acids, carbohydrates)into the aqueous phase of the buffer in which the microbe was disrupted,and the formation of aggregates of insoluble cellular materials. Theconditions for solubilization preferably result in the aggregation ofSRPs and/or porins into insoluble aggregates that are large enough toallow easy isolation by, for instance, centrifugation. The ability toproduce insoluble aggregates was unexpected, and provides for aneconomical way to isolate SRPs and porins.

[0051] Preferably, the sarcosine is added such that the final ratio ofsarcosine to gram weight of disrupted microbe is between about 0.8 gramsarcosine per about 4.5 grams pellet mass and about 1.2 grams sarcosineper about 4.5 grams pellet mass, preferably, about 1.0 gram sarcosineper about 4.5 grams pellet mass. The solubilization of the microbe maybe measured by methods that are routine and known to the art, including,for instance, changes in optical density. Typically, a disrupted microbeis allowed to solubilize until the percent transmitance at about 540 nmis between about 25% and about 30%. Preferably, the solubilization isallowed to occur for at least about 24 hours, more preferably, at leastabout 48 hours, most preferably, at least about 60 hours. Thetemperature during disruption is typically kept low, preferably at about4° C.

[0052] The insoluble aggregates that include the SRPs and porins may beisolated by methods that are routine and known to the art. Preferably,the insoluble aggregates are isolated by centrifugation. Typically,centrifugation of outer membrane polypeptides that are insoluble indetergents requires centrifugal forces of at least 50,000×g, typicallyabout 100,000×g. The use of such centrifugal forces requires the use ofultracentrifuges, and scale-up to process large volumes of sample isoften difficult and not economical with these types of centrifuges.Surprisingly and unexpectedly, the methods described herein provide forthe production of insoluble aggregates large enough to allow the use ofsignificantly lower centrifugal forces (for instance, about 46,000×g).Methods for processing large volumes at these lower centrifugal forcesare available and known to the art. Thus, the insoluble aggregates canbe isolated at a significantly lower cost.

[0053] Optionally and preferably, the sarcosine is removed from theisolated SRPs and porins. Methods for removing sarcosine from theisolated polypeptides are known to the art, and include, for instance,diafiltration, precipitation, hydrophobic, ion-exchange, and/or affinitychromatography, and ultra filtration and washing the polypeptides inalcohol by diafiltration. After isolation, the polypeptides suspended inbuffer and stored at low temperature, for instance, −20° C. or below.

[0054] Another unexpected observation was that this method for obtainingSRPs and proins from a gram negative microbe also resulted in SRPs andporins containing low amounts of LPS. LPS is a potent immunostimulant,and when present in compositions that are administered to animals,especially mammals, can result in decreases in certain performancecharacteristics, and/or injection site reactions that can result in thedowngrading of carcasses due to scaring or blamishes of tissue at theinjection site. The ability to isolate SRPs and porins with low amountsof LPS results in decreased economic losses associated withadministration of preparations from gram negative microbes. Thedecreased amount of LPS results in fewer condemned and/or downgradedcarcasses at slaughter, and fewer decreases in performancecharacteristics.

[0055] SRPs may also be isolated from gram positive microbes usingmethods that are known to the art. The isolation of SRPs from grampositive microbes can be accomplished as described in, for instance,Hussain, et al. Infect. Immun., 67, 6688-6690 (1999); Trivier, et al.,FEMS Microbiol. Lett., 127, 195-199 (1995); Heinrichs, et al., J.Bacteriol., 181, 1436-1443 (1999).

[0056] Methods of use

[0057] An aspect of the present invention is further directed to methodsof using the compositions of the present invention. The methods includeadministering to an animal an effective amount of a composition of thepresent invention. Preferably, the composition includes LPS at aconcentration of, in increasing order of preference, no greater thanabout 10.0 endotoxin units per milliliter (EU/ml), no greater than about5.0 EU/ml, no greater than about 1.0 EU/ml, no greater than about 0.5EU/ml, no greater than about 0.1 EU/ml, most preferably, no greater thanabout 0.05 EU/ml. Preferably, the composition further includes apharmaceutically acceptable carrier. The animal can be, for instance,avian (including, for instance, chickens or turkeys), bovine (including,for instance, cattle), caprine (including, for instance, goats), ovine(including, for instance, sheep), porcine (including, for instance,swine), Bison (including, for instance, buffalo), companion animals(including, for instance, horses), members of the family Cervidae(including, for instance, deer, elk, moose, caribou and reindeer), andhumans.

[0058] In some aspects, the methods may further include additionaladministrations (e.g., one or more booster administrations) of thecomposition to the animal to enhance or stimulate a secondary immuneresponse. A booster can be administered at about 1 week to about 8weeks, preferably about 2 to about 4 weeks, after the firstadministration of the composition. Subsequent boosters can beadministered one, two, three, four, or more times annually. Withoutintending to be limited by theory, it is expected that annual boosterswill not be necessary, as an animal will be challenged in the field byexposure to microbes expressing SRPs and/or porins having epitopes thatare identical to or structurally related to epitopes present on the SRPsand/or porins of the composition administered to the animal.

[0059] In one aspect, the invention is directed to methods for inducingthe production of antibody in an animal. The antibody produced includesantibody that specifically binds at least one polypeptide (an SRP and/ora porin) present in the composition. In this aspect of the invention, an“effective amount” is an amount effective to result in the production ofantibody in the animal. Methods for determining whether an animal hasproduced antibodies that specifically bind polypeptides present in acomposition of the present invention can be determined as describedherein.

[0060] The method may be used to produce antibody that specificallybinds polypeptides, preferably, SRPs and/or porins, present on thesurface of a microbe other than the microbe from which the SRPs andporins of the composition were isolated. As discussed herein, SRPs andporins typically include epitopes that are conserved in the SRPs andporins of different species and different genera of microbes.Accordingly, antibody produced using SRPs and porins from one microbeare expected to bind to SRPs and/or porins present on other microbes(see, for instance, Examples 8 and 10) and provide broad spectrumprotection against gram positive and gram negative organisms. Examplesof gram positive microbes to which the antibody specifically binds aremembers of the family Micrococcaceae, members of the familyDeinococcaceae, or other gram positive microbes as described in thesection “Compositions.” Preferably, gram positive microbes to which theantibody binds are. Staphylococcus aureus, Streptococcus agalactiae,Streptococcus uberis, Streptococcus bovis and Streptococcus dysgalatiae,Streptococcus zooepidemicus, and Streptococcus equi, most preferably,Staphylococcus aureus. Examples of gram negative microbes to which theantibody specifically binds are enteropathogens, more preferably,members of the family Enterobacteriaceae, even more preferably, membersof the Enterobacteriaceae tribes Escherichieae or Salmonelleae, asdescribed in the section “Compositions.” Most preferably, gram negativemicrobes to which the antibody specifically binds are Salmonella spp.and E. coli.

[0061] In an alternative aspect, methods for inducing the production ofantibody in an animal include administering a composition prepared froma whole cell preparation. According to this embodiment, the whole cellpreparation can be prepared from, for example, a modified Escherichiacoli such as a virulent R-mutant, as for example, E. coli J5(commercially available from ATCC as ATCC #43745; described by Overbecket al., J. Clin. Microbiol., 25, 1009-1013 (1987)), or Salmonellaminnesota (commercially available from ATCC as ATCC number #49284; asdescribed by Sanderson et al., J. Bacteriol., 119, 753-759, 760-764(1974)) that lack outer oligosaccharide side chains of LPS. In anon-immunized animal outer oligosaccharide side chains tend to mask SRPson the cell membrane in such a way that the immune system does notrecognize the SRPs and production of anti-SRP antibody titers aredepressed. Thus, to enhance the immune stimulating capability of animmunizing composition made with intact bacterial cells to elicit ananti-SRP immune response, the cell membrane can be chemically altered toeliminate the interfering oligosaccharide side chains or a mutantorganism such as the E. coli J5 organism discussed above can be used.Chemically modified cells or mutants are then grown underiron-restriction conditions to enhance SRP production as described in,for example, U.S. Pat. No. 6,027,736.

[0062] In another aspect, the present invention is directed to methodsfor treating certain conditions in animals that may be caused by, orassociated with, a microbe. Such conditions include, for instance, gramnegative microbial infections and gram positive microbial infections.Examples of conditions caused by microbial infections include mastitis,fecal shedding of a microbe, metritis, strangles, intrauterineinfections, odema disease, enteritis, chronic reproductive infections,laminitis, and acute or chronic Chlamydiosis, Colibacillosis,Ehrlichiosis, Leptospirosis, Pasteurellosis, Pseudotuberculosis,Salmonellosis. Examples of conditions that may be caused by microbialinfections include performance characteristics such as decreased milkproduction, high somatic cell counts, and weight loss. Treatment ofthese conditions can be prophylactic or, alternatively, can be initiatedafter the development of a condition described herein. Treatment that isprophylactic, for instance, initiated before a subject manifestssymptoms of a condition caused by a microbe, is referred to herein astreatment of a subject that is “at risk” of developing the condition.Typically, an animal “at risk” of developing a condition is an animalpresent in an area where the condition has been diagnosed and/or islikely to be exposed to a microbe causing the condition. Accordingly,administration of a composition can be performed before, during, orafter the occurrence of the conditions described herein. Treatmentinitiated after the development of a condition may result in decreasingthe severity of the symptoms of one of the conditions, or completelyremoving the symptoms. Preferably, administration of a compound isperformed before the occurrence of the conditions described herein. Inthis aspect of the invention, an “effective amount” is an amounteffective to prevent the manifestation of symptoms of a disease,decrease the severity of the symptoms of a disease, and/or completelyremove the symptoms. The potency of a composition of the presentinvention can be tested according to standard methods established by 9CFR § 113. For instance, 9 CFR § 113.120(c) and 9 CFR § 113.123(c)describe standard methods for determining the potency of the compositionagainst a standard reference bacterin of Salmonella typhimurium andSalmonella dublin, respectively. Methods for determining whether ananimal has the conditions disclosed herein and symptoms associated withthe conditions are routine and known to the art.

[0063] In one aspect the invention is also directed to treating a gramnegative microbial infection in an animal, and/or a gram positiveinfection in an animal. The method includes administering an effectiveamount of the composition of the present invention to an animal havingor at risk of having a gram positive or a gram negative infection, anddetermining whether at least one symptom of infection is mastitis isreduced.

[0064] In another aspect, the invention provides for treatment ofmastitis in milk producing animals, such as cattle. The method includesadministering an effective amount of the composition of the presentinvention to a milk producing animal having or at risk of havingmastitis, and determining whether at least one symptom of mastitis isreduced. Mastitis refers to inflammation of the mammary gland. Physical,chemical and usually bacteriological changes in the milk andpathological changes in the glandular tissue characterize it. Theseglandular changes often result in a number of symptomatic conditionssuch as, discoloration of the milk, the presence of clots and thepresence of large numbers of leukocytes. Clinically, mastitis is seen asswelling, heat, pain and induration in the mammary gland often resultingin deformation of the udder. In many cases the diagnosis of subclinicalinfections has come to depend largely on indirect tests which depend onthe leukocyte content of the milk or somatic cell count (SCC). The mostcommon organisms that infect the udder are classified into two groups:I) contagious pathogens and 2) environmental pathogens. Examples ofcontagious pathogens include, for instance, Staphylococcus aureus andStreptococcus agalactiae. Examples of environmental pathogens includethe coliforms such as, Escherichia coli, Klebsiella pneumoniae,Klebsiella oxytoca, Enterococcus faecium, Enterococcus faecalis,Enterobacter aerogenes, and Streptococci such as S. uberis, S. bovis andS. dysgalactiae. Examples of other gram negative bacteria which maycause mastitis include, Aerobacter spp., Bacteroides spp., Campylobacterspp., Citrobacter spp., Enterobacter spp., Erwinia spp., Escherichiaspp., Fusobacaterium spp., Klebsiella spp., Leptospira spp., Mycoplasmaspp., Pasteurella spp., Providencia spp., Pseudomonas spp., Proteusspp., Serratia spp., Salmonella spp., and Yersinia spp. Preferably,administration of the composition of the present invention will treatmastitis caused by a gram negative microbe or a gram positive microbe.Preferably, mastitis-causing gram positive microbes that can be treatedusing the present invention are members of the family Micrococcaceae,members of the family Deinococcaceae, or other gram positive microbes asdescribed in the section “Compositions.” More preferably, gram positivemicrobes are Staphylococcus aureus, Streptococcus agalactiae,Streptococcus uberis, Streptococcus bovis and Streptococcus dysgalatiaeand Streptococcus equi, most preferably, Staphylococcus aureus.Preferably, mastitis-causing gram negative microbes that can be treatedusing the present invention are enteropathogens, more preferably,members of the family Enterobacteriaceae, even more preferably, membersof the Enterobacteriaceae tribes Escherichieae or Salmonelleae, asdescribed in the section “Compositions.” Most preferably, gram negativemicrobes are Salmonella spp. and E. coli.

[0065] In yet another aspect, the invention provides for treatment ofmetritis in an animal, preferably in cattle. The method includesadministering an effective amount of the composition of the presentinvention to an animal having or at risk of having metritis, anddetermining whether at least one symptom of metritis is reduced.Metritis is an inflammation of the uterus after calving and is oftencaused by a retained placenta. Subclinical metritis in an animal isoften indicative of decreased performance characteristics, including,for instance, lower milk production, decreased fertility and weightloss, of the animal.

[0066] In another aspect, the invention is directed to a method fortreating high somatic cell counts in an animal's milk, preferably, acow. The method includes administering an effective amount of thecomposition of the present invention to a milk producing animal havingor at risk of having high somatic cell counts, and determining whetherthe somatic cell count in milk obtained from the animal contains reducedsomatic cell counts compared to milk obtained from the animal beforereceiving the composition. In another aspect the invention is directedto a method for reducing somatic cell counts in an animal's milk.Surprisingly and unexpectedly, decreases in somatic cell counts inanimals receiving SRPs and porins from Salmonella did not appear to berelated to clinical disease caused by Salmonella (see results section ofExample 7, and Example 8). Somatic cell count (SCC) is a commonly usedmeasure of milk quality. Somatic cells include leucocytes of the animal,and are typically present at low levels in normal milk. High levels ofsomatic cells in milk, for instance, at least about 250,000 cells permilliliter of milk, preferably, at least about 400,000 cells permilliliter of milk, indicate reduced milk quality. High levels ofsomatic cells in milk may be indicative of infection (mastitis), but mayalso be unassociated with infection (see Example 8). SCC is monitored,typically by milk processing plants, using methods that are routine tothe art. In one aspect, the invention is particularly advantageous forreducing somatic cell counts of milk produced by milk producing animalsinfected with a microbe from the families Acholeplasmataceae,Bacteroidaceae, Enterobacteriaceae, Leptospiraceae, Micrococcaceae,Mycoplasmataceae, Mycobacteriaceae, Neisseriaceae, Pasteurellaceae,Pseudomonadaceae, Spirochaetaceae, or Vibronaceae. Preferably, the SCCis reduced to, in increasing order of preference, less than about750,000 cells/ml, less than about 600,000 cells/ml, less than about400,000 cells/ml, most preferably, less than about 250,000 cells/ml.Gram positive microbes causing increased SCC that can be treated usingthe present method are members of the family Micrococcaceae, members ofthe family Deinococcaceae, or other gram positive microbes as describedin the section entitled “Compositions.” Preferably, gram positivemicrobes are Staphylococcus aureus, Streptococcus agalactiae,Streptococcus uberis, Streptococcus bovis and Streptococcus dysgalatiaeand Streptococcus equi, most preferably, Staphylococcus aureus. Gramnegative microbes causing increased SCC that can be treated using thepresent method are enteropathogens, more preferably, members of thefamily Enterobacteriaceae, even more preferably, members of theEnterobacteriaceae tribes Escherichieae or Salmonelleae, as described inthe section entitled “Compositions.” Most preferably, gram negativemicrobes to which the antibody specifically binds are Salmonella spp.and E. coli.

[0067] In another aspect, the invention is directed to treating low milkproduction by a milk producing animal, preferably, a cow. The methodincludes administering an effective amount of the composition of thepresent invention to a milk producing animal having or at risk of havinga low milk production, and determining whether milk production by theanimal is increased compared to milk production by the animal beforereceiving the composition. In another aspect the invention is directedto a method for increasing milk production in a milk producing animal,preferably, a cow. The method includes administering a composition ofthe present invention to a milk producing animal, and determiningwhether milk production by the animal is increased compared to milkproduction by the animal before receiving the composition. Preferably,the milk production by a milk producing animal after administration ofcomposition of the present invention is increased by at least about 1%,more preferably, by at least about 3%, most preferably, by at leastabout 6%. Preferably, milk production by a cow is determined beforeadministration and about 2 weeks, more preferably, about 8 weeks, mostpreferably, about 16 weeks after administration of the composition.

[0068] In yet another aspect, the invention is directed to treatingintestinal colonization by a microbe, preferably, an enteropathogen.Intestinal colonization by an enteropathogen is typically determined bymeasuring fecal shedding of a microbe by the animal. The method fortreating intestinal colonization by an enteropathogen includesadministering an effective amount of the composition of the presentinvention to an animal having or at risk of having fecal shedding of anenteropathogen, and determining whether the fecal shedding of anenteropathogen is decreased compared to the fecal shedding of themicrobe by the animal before receiving the composition. Fecal sheddingmay be measured by methods routine and known to the art. Many of theanimals infected with an enteropathogen, for instance, Salmonella spp.or E. coli, will shed the microbe in their feces or body excretions.When the microbe is Salmonella, this may serve as a source for chronicSalmonellosis in the herd. Preferably, the microbe is E. coli or aSalmonella spp., more preferably, a Salmonella spp. Preferably, themicrobe includes a polypeptide (for instance, an SRP and/or a porin)that include an epitope that is structurally related to an epitopepresent on an SRP and/or a porin present in the composition administeredto the animal. Preferably, the level of fecal shedding is reduced byabout 10-fold, more preferably, by about 100-fold, even more preferably,by about 1,000-fold. Most preferably, the level of fecal shedding of anenteropathogen is reduced such that the enteropathogen is no longerdetectable.

[0069] The present invention is also directed to methods of increasingmilk quality. Indicators of low milk quality include, for instance,somatic cell counts of at least about 250,000 cells per milliliter ofmilk, preferably, at least about 400,000 cells per milliliter of milk,and microbial contamination of milk. The method includes administeringan effective amount of the composition of the present invention to ananimal, and determining whether the quality of milk from a milkproducing animal is increased compared to the milk quality of the milkproducing animal before receiving the composition. Without intending tobe limited by theory, milk produced by these animal results in thepresence of antibody directed to SRPs and porins in the milk, and theseantibodies will decrease the ability of microbes having cross-reactiveSRPs and/or cross-reactive porins to grow in the milk.

[0070] A composition of the invention can be used to provide for activeor passive immunization against bacterial infection. Generally, thecomposition can be administered to an animal to provide activeimmunization. However, the composition can also be used to induceproduction of immune products, such as antibodies, which can becollected from the producing animal and administered to another animalto provide passive immunity. Immune components, such as antibodies, canbe collected to prepare antibody compositions from serum, plasma, blood,colostrum, etc. for passive immunization therapies. Antibodycompositions comprising monoclonal antibodies and/or anti-idiotypes canalso be prepared using known methods. Passive antibody compositions andfragments thereof, e.g., scFv, Fab, F(ab′)₂ or Fv or other modifiedforms thereof, may be administered to a recipient in the form of serum,plasma, blood, colostrum, and the like. However, the antibodies may alsobe isolated from serum, plasma, blood, colostrum, and the like, usingknown methods and spray dried or lyophilized for later use in aconcentrated or reconstituted form. Passive immunizing preparations maybe particularly advantageous for treatment of acute systemic illness, orpassive immunization of young animals that failed to receive adequatelevels of passive immunity through maternal colostrum.

[0071] Another aspect of the present invention provides methods fordetecting antibody that specifically binds polypeptides of thecompositions of the present invention. These methods are useful in, forinstance, detecting whether an animal has antibody that specificallybind polypeptides of the compositions of the present invention, anddiagnosing whether an animal may have a condition caused by a microbeexpressing SRPs and/or porins of the compositions described herein.Preferably, such diagnostic systems are in kit form. The methods includecontacting an antibody with a preparation that includes polypeptidespresent in a composition of the present invention to result in amixture. Preferably, the antibody is present in a biological sample,more preferably blood, milk, or colostrum. The method further includesincubating the mixture under conditions to allow the antibody tospecifically bind the polypeptide to form a polypeptide:antibodycomplex. As used herein, the term “polypeptide:antibody complex” refersto the complex that results when an antibody specifically binds to apolypeptide. The preparation that includes the polypeptides present in acomposition of the present invention may also include reagents, forinstance a buffer, that provide conditions appropriate for the formationof the polypeptide:antibody complex. The polypeptide:antibody complex isthen detected. The detection of antibodies is known in the art and caninclude, for instance, immunofluorescence and peroxidase.

[0072] The methods for detecting the presence of antibodies thatspecifically bind to polypeptides of the compositions of the presentinvention can be used in various formats that have been used to detectantibody, including radioimmunoassay and enzyme-linked immunosorbentassay.

[0073] The present invention also provides a kit for detecting antibodythat specifically binds polypeptides of the compositions of the presentinvention. The kit includes at least two SRPs and at least two porins ina suitable packaging material in an amount sufficient for at least oneassay. Optionally, other reagents such as buffers and solutions neededto practice the invention are also included. Instructions for use of thepackaged polypeptides are also typically included.

[0074] As used herein, the phrase “packaging material” refers to one ormore physical structures used to house the contents of the kit. Thepackaging material is constructed by wellknown methods, preferably toprovide a sterile, contaminant-free environment. The packaging materialhas a label which indicates that the polypeptides can be used fordetecting SRPs and/or porins. In addition, the packaging materialcontains instructions indicating how the materials within the kit areemployed to detect SRPs and porins. As used herein, the term “package”refers to a solid matrix or material such as glass, plastic, paper,foil, and the like, capable of holding within fixed limits thepolypeptides. Thus, for example, a package can be a microtiter platewell to which microgram quantities of polypeptides have been affixed.“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

[0075] The present invention is illustrated by the following examples.It is to be understood that the particular examples, materials, amounts,and procedures are to be interpreted broadly in accordance with thescope and spirit of the invention as set forth herein.

EXAMPLES

[0076] Compositions including siderophore receptor proteins and porinsfrom Salmonella was evaluated for efficacy against a virulent challengein mice and for the control of Salmonellosis in commercial dairy andfeed lot cattle. The efficacy of the composition was evaluated bycollecting data on the following parameters: first the potency of theimmunizing composition was evaluated against a live virulent challengein mice, and secondly the efficacy was evaluated in commercial dairy andfeed lot cattle by examining the serological response to vaccination,elimination of Salmonella as examined by fecal shedding, reduction inmorbidity and mortality, reduction of somatic cells in milk, total milkproduction, and examination of injections sites after each vaccination.

Example 1 Production and Isolation of Siderophore Receptor Proteins andPorins

[0077] Gram negative bacteria belonging to the familiesEnterobacteriaceae and Pseudomonadaceae, as well as other gram negativebacteria can be grown under controlled fermentation conditions so as toexpress siderophore receptor proteins and porins, and optionally, ironregulated proteins, on the outer membrane. The bacteria can be harvestedby conventional methods and the outer membrane proteins can then beisolated and used as immunogens in a vaccine composition described indetail in the following example.

[0078]Salmonella dublin was isolated from Holstein steers in acommercial feed lot showing clinical signs of Salmonellosis, anddesignated MS010207. The isolate was serotyped by the Minnesota PoultryTesting Laboratory, (Willmar, Minn.). A master seed stock of theorganism was prepared by inoculating 100 ml of Tryptic Soy Broth (DifcoLaboratories, Detroit, Mich.) containing 50 micrograms per milliliter(μg/ml) of 2,2-dipyridyl (Sigma-Aldrich St. Louis, Mo.). The culture wasgrown while stirring at 200 rpm for 6 hours at 37° C. The bacteria werecollected by centrifugation at 10,000×g. The bacterial pellet wasresuspended in 20 ml physiological saline (0.85%) containing 20%glycerol. The bacterial suspension was sterilely dispensed into 20-2 mlcryogenic vials and stored at −90° C. The master seed was expanded intoa working seed that was then used for the production of siderophorereceptor proteins and porins. A large-scale production process wasdeveloped involving fermentation, bacterial harvest, disruption,solubilization, concentration, diafiltration, and isolation of finalproduct.

[0079] Fermentation

[0080] A cryogenic vial of the working seed (1 ml at 109 CFU/ml) wasused to inoculate 500 ml of Tryptic Soy Broth (TSB) without dextrose(Difco) pre-warmed to 37° C. containing 50 micrograms 2,2-dipyridyl(Sigma), 2.7 grams BiTek yeast extract (Difco) and glycerol (3%vol/vol). The culture was incubated at 37° C. for 12 hours whilestirring at 200 rpm at which time was inoculated into 2 liters of theabove media and allowed to grow for an additional 4 hours at 37° C. Thisculture was used to inoculate a 20-liter Virtis bench-top fermentor,(Virtis, Gardiner, N.Y.) charged with 13 liters of the above-describedmedia. The pH was held constant between 6.9 and 7.1 by automatictitration with 30% NaOH and 10% HCL. The stirring speed was adjusted at400 rev/minute, and the culture aerated with 11 liters air/minute at 37°C. Foaming was controlled automatically by the addition of 11 mldefoamer (Mazu DF 204 Chem/Serv, Minneapolis, Minn.). The culture wasallowed to grow continuously at these conditions for 4 hours at whichtime was sterilely pumped into a 150-liter fermentor (W. B. Moore,Easton, Pa.). The fermentor was charged with 115 liters tryptic soybroth without dextrose (3,750.0 grams), BiTek yeast extract (625 grams),glycerol (3750 ml), 2,2-dypyrdyl (3.13 grams) and Mazu DF 204 defoamer(100 ml). The parameters of the fermentation were as follows: dissolvedoxygen (DO) was maintained at 30% +/−10% by increasing agitation to 220rev/minute sparged with 60 liters of air/minute and 10 pounds per squareinch (psi) back pressure. The pH was held constant between 6.9 and 7.1by automatic titration with 30% NaOH and 10% HCL. The temperature wasmaintained at 37° C. At hour 4.5 (OD₅₄₀ 8-9) of the fermentation theculture was supplemented with additional nutrients by feeding 7 litersof media containing 1,875 grams TSB without dextrose, 313 grams yeastextract 3.13 grams 2,2-dipyridyl and 1,875 ml of glycerol. The rate offeed was adjusted to 29 ml/minute while increasing agitation to 675 rpm.At the end of the feed (hour 8.5) the fermentation was allowed tocontinue for an additional three hours at which point the fermentationwas terminated by lowing the temperature of the fermentor to 10° C.(OD₅₄₀ 3540 at a 1:100 dilution). The culture was sterilely transferredto a 200-liter tank (LEE Process Systems and Equipment model 2000LDBT)in preparation for harvest.

[0081] Harvest

[0082] The bacterial fermentation was concentrated and washed using aPall Filtron Tangential Flow Maxiset-25 (Pall Filtron Corporation,Northboro, Mass.) equipped with two 30 ft² Alpha 300-K open channelfilters, catalog No. AS300C5, (Pall Filtron) connected to a WaukeshaModel U-60 feed pump (Waukesha Cherry-Burrell, Delevan, Wis.) Theoriginal culture volume of 125 liters was reduced to 25 liters (2.5liters/minute) using a filter inlet pressure of 15 psi and a retentatepressure of 0 psi. The bacterial retentate was adjusted back up to 50liters using physiological saline (0.85%) and then concentrated again to15 liters to help remove any contaminating exogenous proteins, etc. Theretentate (15 liters) was adjusted to 35 liters using sterile OsmoticShock Buffer (OMS) containing 7.26 grams/liter Tris-base and 0.93grams/liter EDTA adjusted to a pH of 8.5. The EDTA in the OMS serves toremove much of LPS from the cell wall, while the elevated pH preventsmuch of the proteolytic degradation after freezing and disruption.Protease inhibitors may be used instead of, or in addition to, anelevated pH. The retentate was mixed thoroughly while in the 200-litertank using a bottom mount magnetically driven mixer. The retentate wassterilely dispensed (3.5 liters) into sterile 4 liter Nalgene containersNo. 2122 and placed into a −20° C. freezer for storage. Freezing thebacterial pellet serves to weaken the cell wall structure makingdownstream disruption more efficient. The pellet mass was calculated bycentrifuging 30 ml samples of the fermented culture and final harvest.Briefly, pre-weighted 50 ml Nalgene conical tubes were centrifuged at39,000×g for 90 minutes in a Beckman J2-21 centrifuge using a JA-21rotor (Beckman Instruments, Palo Alto Calif.). At the end of the run,the supernate was poured off and the tubes were weighed again. Thepellet mass was calculated for each stage. The fermentation processyielded a wet pellet mass of 9.0 kilograms.

[0083] Disruption (Homogenization)

[0084] Twenty kilograms of frozen bacterial cell slurry in OMS werethawed at 4° C. (20 kg of pellet mass). The liquid culture suspensionfrom each container was aseptically aspirated into a steam in place 250liter jacketed process tank (Lee, Model 259LU) with a top mounted mixer(Eastern, Model TME-1/2, EMI Incorporated, Clinton, Conn.) containing222 liters OMS pH 8.5 containing 0.1 grams thimerosal/liter aspreservative. The volume of OMS was determined by dividing the pelletmass (in grams) by 900 and then multiplying the result by 10 to get thehomogenizing volume in liters (gram pellet mass/900×10=litershomogenizing volume). The bulk bacterial suspension was chilled to 4° C.with continuous mixing for 18 hours at 200 rpm at which time wasdisrupted by homogenization. Briefly, the 250 liter tank containing thebacterial suspension was connected to a model 12.51H Rannie Homogenizer,(APV Systems, Rosemont, Ill.). A second 250 liter jacketed process tank(empty) was connected to the homogenizer such that the fluid in theprocess tank could be passed through the homogenizer, into the emptytank and back again, allowing for multiple homogenizing passes whilestill maintaining a closed system. The temperature during homogenizationwas kept at 4° C. At the start of each pass, fluid was circulated at 70psi via a Waukesha model 10DO pump (Waukesha) through the homogenizer(160 gallons/hour) and back to the tank of origin, while the homogenizerpressure was adjusted to 13,500 psi. Prior to the first pass, twopre-homogenizing samples were withdrawn from the homogenizer toestablish a baseline for determining the degree of disruption andmonitoring of pH. The degree of disruption was monitored bytransmittance (% T at 540 nm at 1:100 dilution) compared to thenon-homogenized sample. The number of passes through the homogenizer wasstandardized for different organisms based on the integrity of the cellwall and variation in the degree of disruption, which had a directcorrelation in the efficiency of solubilization and quality of endproduct. For example, the disruption of Salmonella passed three timesthrough the homogenizer gave a final percent transmittance between78-83% T at a 1:100 dilution. E. coli having the same pellet mass andstarting OD gave a % T of 86-91% (at a 1:100 dilution) after the thirdpass. It has been observed that bacteria differ in their cell wallintegrity and vary in their capacity of disruption under identicalcondition. This variation can effect the degree and efficiency ofsolubilization and recovery of SRPs and porins from the outer membrane.In general, cells were passed through the homoginizer until thetransmittance did not increase after an additional pass.

[0085] After homogenization, Sodium Lauroyl Sarcosinate (Hamptosyl L-30,Chem/Serv) was aseptically added to the homogenized bacterial suspensionfor solubilization. The amount of Sarcosine (30%) added equaled 0.0664times the solubilizing volume, in liters, (1.0 gram sarcosine/4.5 gramspellet mass). The tank was removed from the homogenizer and put onto achiller loop at 4° C. and mixed at 240 rpm for 60-70 hours. This timeperiod was important for complete solubilization. It was discovered thatincreasing the solubilization time in OMS at an elevated pH (8.0-8.5)that the SRPs and porins aggregated together forming large insolubleaggregates that were easily removed by centrifugation. The optimal ODafter solubilization was usually between 25-30% T at 540 nm.

[0086] Protein Harvest

[0087] The aggregated siderophore receptor proteins and porins withinthe solubilized process fluid were collected by centrifugation using T-1Sharples, (Alfa Laval Seperations, Warminster, Pa.). Briefly, the tankof solubilized homogenate was fed into six Sharples with a feed rate of250 ml/minute at 17 psi at a centrifugal force of 46,000×g. The effluentwas collected into a second 250 liter jacketed process tank through aclosed sterile loop allowing for multiple passes through the centrifugeswhile maintaining a closed system. The temperature during centrifugationwas kept at 4° C. The solubilized homogenate was passed 8 times acrossthe centrifuges. Fifty percent of the protein was collected after thesecond pass, at which point, the solubilized fluid was concentrated to ⅓of its original volume, which shortened the process time for the next 6passes. Briefly, the solubilized homogenate tank was asepticallydisconnected from the centrifuges and connected to a Millipore PelliconTangential Flow Filter assembly (Millipore Corporation, Bedford, Mass.),equipped with a 25 ft² screen-channel series Alpha 10K Centrasettefilter (Pall Filtron) connected to a Waukesha Model U30 feed pump forconcentration. After concentration, centrifugation was continued untilthe process was completed. Protein was collected after each pass. Theprotein was collected, resuspended and dispensed in 50 litersTris-buffer pH 8.5 containing 0.3% formulin (Sigma) as preservative.

[0088] Diafiltration

[0089] The protein suspension was washed by diafiltration at 4° C. toremove any contaminating sarcosine that may be bound to the protein.Briefly, the 50 liters of protein was sterilely aspirated into a 200liter process tank containing 50 liters sterile Tris-buffer, pH 8.5equipped with a bottom mount Dayton mixer, Model 2Z846 (Dayton Electric,Chicago, Ill.) rotating at 125 rev/minute. The process tank wassterilely connected to a Millipore Pellicon Tangential Flow Filterassembly (Millipore Corporation), equipped with a 25 ft² screen-channelseries Alpha 10K Centrasette filter (Pall Filtron) connected to aWaukesha Model U30 feed pump. The 100 liter protein solution wasconcentrated by filtration to a target volume of 5.45 times the proteinpellet mass at which point Tris-buffer pH 7.4 containing 5% isopropylalcohol was slowly added to the concentrate from a second process tank.Isopropyl alcohol causes a slight unfolding of the protein structureallowing for the removal of bound sarcosine without compromising theimmunogenicity of the protein. Diafiltration continued until the pHstabilized to 7.4 at which point 50 liters Tris-buffer pH 7.4 was slowlyadded by diafiltration to remove residual alcohol. The proteinsuspension was then concentrated to approximately 25 liters. The proteinconcentrate was aseptically dispensed (3.5 liters) into sterile 4 literNalgene containers and placed into a −20° C. freezer for storage.

[0090] This process produces an extremely pure composition of SRPs andporins with almost the complete removal of LPS with very little to nosarcosine residue. The protein was examined by SDS-PAGE for purity andbanding profile, bacterial contamination, residual sarcosine and LPS.The banding profile of the finished product showed consistent patternsas examined by electrophoresis. The composition was tested for sarcosineby the use of a modified agar gel diffusion test in which sheep redblood cells (5%) were incorporated into an agar base (1.5%). Wells werecut into the agar and samples of the finished product along with controlsamples of known concentrations of sarcosine at 0.05, 0.1, 0.2, 0.3,0.4, 0.5., 1.0 and 2.0% were placed into the wells. The gel wasincubated at 25° C. for 24 hours and the degree of hemolysis wasdetermined compared to the controls. The process removes the level ofdetectable sarcosine below 0.05%, which at this concentration showedminimal hemolysis in control samples.

[0091] LPS was removed below the detection level as examined by aLimulus amebocyte lysate (LAL) test available under the tradenameE-TOXATE (Sigma Chemical Co., St. Louis, Mo.).

Example 2 Mouse Vaccination and Challenge Study

[0092] The efficacy of a Salmonella dublin vaccine consisting ofSiderophore receptor proteins (SRPs) and porins was carried out againsta live virulent challenge in mice as described under 9 CFR 113.123.Sixty female CF-i mice obtained from Harlan Breeding Laboratories(Indianapolis, Ind.) weighing 16-22 grams were equally distributed into6 polycarbonate mouse cages (Ancore Corporation, Bellmore, N.Y.)designated as groups 1-6.

[0093] The composition including siderophore receptor proteins andporins was prepared as described in Example 1 from a bovine fieldisolate of Salmonella dublin originating from a herd of Holstein Dairycows showing clinical symptoms of Salmonellosis.

[0094] The SRPs had molecular weights of 89 kDa, 84 kDa, 72 kDa andporins had molecular weights of 38-39 kDa as examined on a 12% SDS-Pagegel. The SRPs and porins in 8.3 ml (6,035 μg/ml) were resuspended into69.2 ml physiological saline (0.85%). The aqueous protein suspension(77.5 ml) was emulsified into 22.5 ml EMULSIGEN, (MVP Laboratories,Ralston, Nebr.) using a]KA Ultra Turrax T-50 homogenizing vessel (IKA,Cincinnati, Ohio) to give a final dose of 125 μg total protein in a 0.25ml injectable volume at a 22.5% vol/vol adjuvant concentration. Themouse dose was adjusted to be equivalent to a field dose of 1,000 μg ata 2 ml volume.

[0095] The potency of the vaccine was tested at four differentconcentrations, non-diluted (Group-1), 1:10 (volume diluent:volumeprotein solution) (Group-2), 1:100 (Group-3), and 1:1000 (Group-4)compared to two control groups; a non-vaccinated challenged group(Group-5) and a non-vaccinated non-challenged group (Group-6).EMULISIGEN was used as the diluent for diluting the stock vaccine at a22.5% concentration prepared in physiological saline. Mice werevaccinated intraperitoneally and revaccinated 14 days after the firstvaccination. The volume administered was 0.25 cc.

[0096] Fourteen days after the second vaccination, mice in groups 1-5were intraperitoneally challenged with 1.7×10⁸ colony forming units(CFU) of a virulent Salmonella dublin isolate. The isolate (IRP SCCSerial) was obtained from The Center of Veterinary Biologics-Laboratory,United States Department of Agriculture, Ames, Iowa. Mortality wasrecorded daily for 2 weeks post-challenge. Table 1 below shows themortality between the vaccinated and non-vaccinated mice followingchallenge. TABLE 1 Mortality of Vaccinated and Non-Vaccinated MiceFollowing Challenge with Salmonella dublin Groups # Mice # Dead Percentmortality (%) Group-1 (non-diluted) 10 0/10 0 Group-2 (1:10) 10 1/10 10Group-3 (1:100) 10 3/10 50 Group-4 (1:1000) 10 5/10 60 Group-5 (non- 1010/10  100 vaccinated/challenged Group-6 (non-vaccinated/non- 10 0/10 0challenged

[0097] Ten (100%) of the non-vaccinated mice (Group-5) died within 14days after challenge (Table 1). In contrast, none of the mice died giventhe non-diluted vaccine of group-1. All dilutions of the test vaccineshowed a high degree of protection as compared to thenon-vaccinated/challenged mice of group-5. None of the mice died ingroup-6 showing no horizontal transmission of the organism betweengroups.

Example 3 Preparation of an Immunizing Composition Derived fromSalmonella bredeney

[0098]Salmonella bredeney was isolated and serotyped from a Minnesotadairy herd having a history of high adult and calf mortality, morbidityand loss of production due to this bacterial strain, and designatedMS010914. SRPs and porins were isolated as described in Example 1. Threehigh molecular weight SRPs, 89 kDa, 84 kDa, and 72 kDa, were observed ona 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-Page) gel. Three additional lower molecular weight iron-regulatedproteins (IRPs) were also isolated at approximately the 37 kDa, 32 kDaand 29 kDa regions. Porins having a molecular weight in the range of38-39 kDa were also purified from the propagated isolates.

[0099] Two compositions were prepared from the SRPs having molecularweights of 89 kDa, 84 kDa, and 72 kDa, the IRPs having molecular weightsof 37 kDa, 32 kDa, and 29 kDa, and porins having molecular weights of38-39 kDa. The target proteins were emulsified in the following vaccineformulations to provide a total dose of about 1,000 μg. In the firstcomposition, referred to as Vac-1,50 ml of antigen (4.35milligram/milliliter (mg/ml)) was slowly added while stirring to 40 mlof 25% aluminum hydroxide (Rehydagel-HPA, Reheis, N.J.) prepared in 270ml physiological saline. The antigen/aluminum hydroxide suspension wasstirred for 24 hours at 4° C. The antigen/alurninum hydroxide suspensionwas then emulsified into 40 ml of EMULSIGEN, to give a final dose of1,000 μg total protein in a 2 ml injectable volume.

[0100] In the second composition, referred to as Vac-2, 217.25 mg of theSRP antigen was mixed into 270 ml of physiological saline. The antigensolution was emulsified into 80 ml of EMULSIGEN to give a final dose of1,000 μg total protein in a 2 ml injectable volume.

Example 4 Pre-Testing of the Immunizing Compositions

[0101] To determine any possible side effects (e.g. reduced milkproduction, adverse tissue reaction, etc.), the vaccines of Example 3were first administered to cattle from various stages of production: 2lactating cows, 2 non-lactating adult cows, and 2 calves. Two days priorto pre-testing, two lactating cows were selected to determine theirdaily milk production. Milk production from each cow was also monitoredat each of the two daily milkings for two consecutive days (48 hours)after vaccination to determine any loss in production due tovaccination. Monitoring was repeated on a single milking from each cowon day 7. The two lactating cows received 2 mls of Vac-1 subcutaneouslyin the neck region. In addition, another 2 non-lactating cows wereadministered 2.0 ml of Vac-2 subcutaneously in the neck region and twocalves were administered 1.0 ml of Vac-1 subcutaneously in the neckregion and the animals monitored for 7 days for any adverse reaction.

[0102] No adverse tissue reactions were observed at any of the injectionsites of the 6 animals given the pre-test vaccines. In addition, therewas no measurable loss in milk production from the lactating cows at 2and 7 days after vaccination.

Example 5 Herd Immunization

[0103] After completion of the study of Example 4, immunizingcompositions of Vac-1 and Vac-2 were administered to the entire herd.The herd consisted of 55 lactating cows, 52 non-lactating cows and 18calves ranging in age from 6 months to 12 months. Lactating cattlereceived 2.0 ml of Vac-1; non-lactating cattle received 2.0 ml of Vac-2;calves less than 12 months of age but older then 6 months received 1.0ml of Vac-1; and calves greater then 12 months of age received 1.0 ml ofVac-2 (see Table 2). All injections were delivered subcutaneously in theneck region. TABLE 2 Schedule of events. STUDY DAY DESCRIPTION OF EVENTSPre-testing Vaccinated 2 lactating cows, 2 non-lactating cows and 2calves. Monitored milk production and adverse reactions. Firstvaccination Vaccinated all lactating and non-lactating cows (2 ml)except for Day 0 calves, under 12 months, gave 1 ml and 2 ml if olderthen 12 months. Collected blood and fecal samples from lactating cows.Week 3 Collected blood and fecal samples from lactating cows, examinedinjection sites. Second vaccination Vaccinated all lactating andnon-lactating cows (2 ml) except for the Week 5 calves, under 12 months,gave 1 ml and 2 ml if older then 12 months. Week 7 Collected blood,fecal samples and examine injection sites. Week 11 Collected blood,fecal samples and examine injection sites. Third vaccination Vaccinatedall lactating and non-lactating cows (2 ml) except for the Week 19calves, under 12 months, gave 1 ml and 2 ml if older then 12 months.Week 21 Collected blood, fecal samples and examine injection sites. Week35 Collected blood and fecal samples Week 44 Collected blood and fecalsamples

[0104] Thirty five days after the first vaccination all animals wereadministered a second dose (booster) subcutaneously in the neck. For thebooster dose, all lactating cows received 2.0 ml of Vac-1, non-lactatingcows received 2 ml Vac-2, calves between 6-12 months of age received 1.0ml of Vac-1, and animals 12 months of age or older received 1.0 ml ofVac-2. The schedule of events is shown in Table 2.

[0105] Based on the lack of reaction and observed safety of theimmunizing compositions, the herd was vaccinated a third time, 19 weeksafter the first vaccination (Table 1). The target proteins wereemulsified into a single formulation used in all cows, referred to hereas Vac-3. Briefly, 300 mg antigen (SRP and porins) was mixed into 250.96ml of physiological saline. The antigen solution was emulsified into 80ml of EMULSIGEN to give a final dose of 1,000 μg total protein at a22.5% EMULSIGEN concentration in a 2 ml injectable volume. All lactatingand non-lactating cows received a 2 ml intramuscular injection whilecalves 6 months of age and older received a 1 ml intramuscularinjection.

Example 6 Blood and Fecal Sample Collection and Somatic Cell Counts

[0106] Blood samples were collected from twenty lactating cows on theinitial day of immunization (day 0) and again at 3, 7, 11, 21, 35 and 44weeks after the initial immunization. In addition, fecal samples weretaken from all lactating cows on the day of immunization (day 0) andagain at 3, 7, 11, 21, 35 and 44-weeks after immunization (Table 2).

[0107] All blood was collected in sterile 13×75 millimeter (mm)vacutainer collection tubes, brand SST No. 369783, (Becton Dickinson,Franklin Lakes, N.J.). After clotting, the blood tubes were centrifugedat 800×g for 30 minutes and frozen at −20° C. until analysis.

[0108] Individual fecal samples were taken aseptically by rectalextraction using sterile shoulder length gloves and placed in sterilewhirl pack bags. Ten grams of feces from each sample was placed into 90ml of Tetrathionate broth (Difco) and incubated at 37° C. for 24 hours.Each sample was plated onto Bismuth sulfite, Brilliant green and XLDagar (Difco) as a differential selective media to identify the presenceof Salmonella. All suspect isolates were confirmed to be Salmonellausing Salmonella 0 antiserum (poly A-I and Vi) with a slideagglutination test. Briefly, a colony is removed from a plate and mixedin a drop of poly 0 antiserum. This is mixed for about 30 seconds if itagglutinates it's a confirmed suspect. Confirmed Salmonella isolateswere sent to the Minnesota Poultry Testing Laboratory (MPTL), Willmar,Minn., for serotyping.

[0109] Somatic cell counts were per milliliter of milk were conducted bythe Dairy Herd Improvement Association (DHIA, Buffalo, Minn.) usingstandard methods. The somatic cells counted were the white blood cellspresent in the milk.

Example 7 Enzyme-Linked Immunosorbent Assay (ELISA)

[0110] An Enzyme-Linked Immunosorbent Assay (ELISA) monitored theserological response to the vaccine. The highly conserved SRPs fromSalmonella bredeney having molecular weights of 89 kDa, 84 kDa, and 72kDa were purified from polyacrylamide gels. Briefly, the correspondingSRP bands (89 kDa, 84 kDa, and 72 kDa) were cut from unstained gelsusing a stained indicator lane for determining band location which wascut away from the original gel and stained. Elution of the protein fromthe macerated gel was carried out according to the manufacturesrecommendation using a model 422 electro-eluter (Bio-Rad, Laboratories,Hercules, Calif.). These proteins were then used as the capture moleculein an indirect ELISA test.

[0111] Polyclonal antiserum was raised against the vaccine compositionof example 4. Briefly, the vaccine composition consisting of SRPs andporins of Salmonella bredeney was inoculated subcutaneously into 2 adultHolstein heifers (2 ml dose at 1000 ug total protein). Each Heiferreceived a total of three vaccinations 21 days apart. Fourteen daysafter the third vaccination 20 ml of blood was collected from the tailvein of vaccinated cows. In addition negative control serum (20 ml) wasobtained from two non-vaccinated cows. The hyperimmune and negativeserum was obtained by centrifugation (800×g) of the clotted blood. Thehyperimmune and control sera was absorbed with killed whole cellbacteria of Salmonella bredeney grown in iron-replete media (BHIcontaining 200 um ferric chloride) for 1 hour at 4° C.

[0112] Twenty milliliters of the positive and negative control sera wasprecipitated for 6 hours using ammonium sulfate (60% saturation),dissolved in 0.02 M phosphate buffer pH 7.0 at 4° C. The precipitate wascollected by centrifugation at 8000×g for twenty minutes. The pellet wasresuspended in 20 ml of 50 mM phosphate buffer pH 7.2 and dialyzed usinga 100,000 MWCO dialysis tubing (Pierce, Rockford, Ill.) against 0.02 MpH 7.2. The dialyzed material was concentrated 10 times using a Diafloultrafiltration apparatus model 8200 with a 50,000 MWCO membrane(Amicon). The positive and negative control dialysate was alquoted into100 ul samples and frozen at −90° C.

[0113] The optimum working concentrations of SRP and conjugate wasdetermined by several checkerboard titrations using the positive andnegative control dialysates. A prediction curve was then established tocalculate SRP ELISA titers at a 1:500 dilution. All subsequent testswere performed at a single serum dilution (1:500) and SRP titers werecalculated from the average of duplicate test absorbance values.

[0114] The ELISA was performed by adding 100 ul of diluted SRP ofSalmonella in 0.05 M carbonate buffer (pH 9.6) to each well of a 96-wellflat bottom, easy wash microtiter plate (Corning, Corning N.Y.). Afterovernight incubation at 4° C., excess SRP was removed and the plate waswashed. All subsequent washing steps were done three times in phosphatebuffered saline (pH 7.4) with 0.05% Tween-20. The plates were blockedfor one hour at 37° C. with 4% fish gelatin (Sigma) in PBS and thenwashed.

[0115] Duplicate serum samples from Example 4 were tested in parallel atsingle-point dilutions using 100 ul/well and incubated for 45 minutes at37° C. The first two rows of each plate contained the negative andpositive control samples while the rest of the plate was used for thetest samples. The plate was incubated for 45 minutes at 37° C. whilestirring at 200 rpm. After washing, 100 ul alkaline phosphataseconjugate (Monoclonal anti-bovine IgG clone BG-18, Sigma) at a 1:15,000dilution was added to each well. After incubation for 45 minutes at 37°C., the plates were washed and 100 ul p-NitroPhenyl Phosphate (pNPP)substrate (Sigma) was added to each well. The substrate was allowed toreact for 2 hours at 37° C. while stirring at 100 rpm. The reaction wasterminated by the addition of 25 ul of 3N NaOH. The absorbence was readat 405 nm.

[0116] Results of Examples 3-7

[0117]FIG. 1 shows the cumulative history of the shedding prevalence ofSalmonella compared to the serological response to vaccination inlactating cows. As described in Example 5, the herd was vaccinated onthe day of the initial immunization (Day 0) and again at 5 and 19 weeksafter the first vaccination. Fecal and blood samples were taken from alllactating cows at 0, 3, 7, 11, 21, 35, and 44 weeks. Briefly, theimmunizing compositions consisting of Vac-1 and Vac-2 were given to allcows (N=125) in the herd on the day of the initial immunization, day 0(Table 1). Only the lactating cows were monitored through theexperimental trial. All lactating cows were subcutaneously given 2 ml ofVac-1. The shedding prevalence of Salmonella in the fecal samples takenfrom the lactating cows (N=55) on day 0 revealed an isolation rate of85.4% (FIG. 1). All of the Salmonella isolates were serotyped and foundto be S. bredeney.

[0118] Within this same time period the somatic cell count as determinedby DHIA was 1,492,000 cells per milliliter of milk (Table 3), thehighest it had ever been in the history of the farm. TABLE 3 The SomaticCell Count (SCC)¹ of Individual Cows Before and After Vaccination SCCbefore/after vaccination² Cow ID SCC × 1000  1 1980/3930  2 9990/3870  31230/3370  4 1240/2090  5 4390/1980  6 5510/1660  7 2090/1550  8 870/1170  9 3020/960  10 2620/950  11 1040/780  12 1330/720  13 120/72014 570/680 15  63/520 16 460/460 17 9990/450  18 3890/360  19 160/350 20570/290 21 7070/250  22 210/240 23 230/220 24  50/210 25 190/190 26540/180 27 970/160 28 1150/130  29 140/120 30  50/120 31 660/120 32450/120 33 380/110 34 220/100 35 350/100 36 70/90 37 890/60  38 700/60 39 100/50  40 40/50 41 140/50  42 210/40  43 70/40 44 80/30 45 90/30 46110/30  47 230/30  48 3200/20  49 20/20 50 50/20 51 40/10 Average SCC1492/585

[0119] Three weeks after the first vaccination, fecal samples taken fromall lactating cows (N=54) revealed no significant change in the sheddingprevalence of Salmonella, which remained at 87%, (FIG. 1). Nevertheless,the somatic cell count dropped to 585,000 cells per milliliter. This isgraphically and numerically depicted in FIG. 2 and table 3 which showsthe DHIA somatic cell count on individual cows before and after thefirst vaccination. There was a 61.0% drop in somatic cell count having adegree of significance of P=0.0068. This highly significant affect wasobserved without improvements in management and/or environmentalchanges. One year after the first vaccination the cumulative 12 monthaverage in somatic cell count was 417,000 cells per milliliter of milk.In contrast, the 12 month average before vaccination was 660,000 somaticcells per milliliter of milk. This was a 37% decrease in the somaticcells after vaccination. It is interesting to speculate that because ofthe conserved nature of these proteins it induced a degree ofcross-protection against other gram negative or gram positive bacteriaresponsible for contagious and/or environmental mastitis.

[0120] The injection sites of all calves and lactating cows wereexamined 14 days after the first vaccination. None of the cows examinedshowed any adverse tissue reaction at the site of injection by physicalexamination. In addition, there was no measurable loss in milkproduction due to vaccination.

[0121] Five weeks after the first vaccination the herd was given abooster (Table 2). Fourteen-days after the second vaccination (Week 7)there was a 21.2% drop in the shedding prevalence of Salmonella with thetotal number of isolations being 35 out of 54 samples taken or, 64.8% ofthe herd positive for Salmonella in contrast to a previous prevalence of86% (FIG. 1). The isolation rate continued to decline and by theeleventh week the shedding prevalence was 47.1% or 24 positive isolatesout of 51 cows sampled. This was a 52.9% reduction in the number ofpositive Salmonella isolations. Physical examination of the injectionsites showed no adverse tissue reaction in any of the calves and/orlactating cows examined. However, the second vaccination resulted inapproximately a 2% drop in milk production that began 24 hours aftervaccination but lasted less than two days. At this point the data showedthe vaccine compositions to be highly tissue compatible with minimalloss in milk production. In addition, the data indicated a directcorrelation between the declining shedding prevalence of Salmonella tothe increasing SRP antibody response.

[0122] To stimulate a higher SRP antibody response the herd wasvaccinated a third time, fourteen weeks after the second vaccination(Week 19, Table 2). The protein concentration of the vaccine remainedthe same (1000 μg/2 cc dose) but the adjuvant (EMULSIGEN) was increasedto 22.5% vol/vol. Blood and fecal samples were taken 14-days aftervaccination (Week 21, Table 2). The shedding prevalence of Salmonelladeclined to 45%, i.e., only 28 cows out of 61 sampled were positive forSalmonella (FIG. 1). All of these samples were serotyped and found to beSalmonella bredeney. At this same time period the injection sites ofeach lactating cow was examined, and less than 5% developed a granulomathat measured approximately 1 centimeter X 1 centimeter. Thesegranulomas resolved within 21 days after injection.

[0123] The cumulative pounds of milk produced before and after the thirdvaccination is shown in FIG. 3. After the third vaccination the drop inmilk production peaked at 6.9%. This loss in production appearedtransient within the herd, lasting less then four days, at which pointthe herd regained normal production. In fact, after the thirdvaccination the average milk production for the remainder of the monthincreased by 1.2% as compared to production before vaccination (FIG. 3).This increase in milk production was consistent and started at thebeginning of the first vaccination. For example, the DHIA rolling herdaverage for 45 cows for the month of December (year 1, beforevaccination) was 16,787 pounds of milk (FIG. 4 and Table 4). The generalhealth and overall performance of the herd increased after eachvaccination. Fourteen days after the third vaccination (December) therolling herd average for 53 cows was 18,047 pounds of milk produced(FIG. 4 and Table 3). This was a 7.0% increase in milk per cow or andaverage of 1,260 pounds per year. In addition, the annual pounds of milkproduced 1 year after vaccination was 965,472 pounds compared to 740,855pounds produced before vaccination. This was a 6% increase in the totalpounds of milk produced. TABLE 4 The Annual Herd Summary From the Onsetof the First Salmonella isolation DHI Rolling Herd Average-Entire HerdYear Sampled (year 1) Year Sampled (year 2) Year Sampled (year 3) Date¹DIM² Milked³ Lbs.⁴ Date DIM Milked Lbs. Date DIM Milked Lbs. Jan 183 4915563 Jan 201 50 16535 Jan 241 55 18703 Feb 170 58 15795 Feb 171 5716258 Feb 260 54 18776 Mar 173 56 15725 Mar 155 62 16310 Mar 251 5418516 Apr 192 53 15643 Apr 163 60 16409 Apr 227 55 18261 May 217 5115584 May 157 63 16421 May 220 52 18068 Jun 229 49 15467 Jun 165 6416574 Jul 244 51 15503 Jul 161 56 16849 Aug 280 51 15841 Aug 182 5617080 N/A N/A N/A N/A Sept 207 56 17329 Oct 300 48 16315 Oct 231 5417570 Nov 291 48 16606 N/A N/A N/A N/A Dec 264 45 16787 Dec 229 53 18047

[0124]FIG. 5 shows the average monthly cost in antibiotic usagecalculated 12 months after the first vaccination compared to 12 monthsbefore vaccination. The average monthly cost in antibiotic usage beforevaccination or during the course of Salmonellosis was $284.65 comparedto $144.05 after vaccination. This was a 51% reduction in the cost ofantibiotics.

[0125] At the onset of the first isolation of Salmonella bredeney(January, year I) and clinical diagnosis from this herd, approximately21 adult cows and 36 calves died of clinical Salmonellosis. Thismortality occurred despite vaccinating the herd three separate timesover a one year period, using a commercial whole cell bacterin ofSalmonella dublin and Salmonella typhimurium. The herd was up to date inall, routine viral and bacterial vaccines. After the first vaccinationwith the composition described in Example 3, mortality and morbidityvirtually ceased. Six months after the first vaccination only threecalves died within this time period. None of the calves that died withinthis time period were diagnosed with Salmonella. There has been nomortality in any of the non-lactating (dry), lactating cattle and/orcalves in this herd, since the first Salmonella vaccination. From thisdata it would appear the vaccine induced a high degree of humoralimmunity against field challenge as well as providing passive immunityto newborn calves. It was also apparent in this field study that as theserological response to vaccination increased, the shedding prevalenceof Salmonella decreased. It is interesting to note that the antibodytiter continued to rise 25 weeks after the third vaccination. Thiscontinued rise in titer could be due to clinical field challenge bySalmonella or other gram negative bacteria expressing these highlyconserved proteins during subclinical infections.

[0126] Vaccination improved the overall health and performance status ofthe herd as observed by the decrease in mortality, decreased somaticcell counts and the increase in milk production. Calf health alsoimproved, as calves were more active at birth, consumed colostrumaggressively and did not develop any significant diarrhea symptoms. Inaddition, there was an observed decrease in clinical metritis in thefresh cows that were brought back into production after calving. Thesecows were vaccinated at dry off and boosted prior to calving.Vaccination appeared to alleviate the incidence of clinical metritisduring the post-calving period. This was initially observed while takingfecal samples for the isolation of Salmonella in that rectal palpationof the uterus could be done at the same time. The incidence of metritisdramatically decreased after vaccination as compared to previous years.

[0127] The vaccine composition proved to be highly tissue compatible.None of the vaccinated cows showed any adverse tissue reaction at thesite of injection or any physical signs of stress such as, depression,lethargy, loss of milk production, etc. The compatibility of the vaccinecomposition is likely due to its purity and lack of contaminatinglipopolysaccharides (LPS). LPS has been shown to be responsible for muchof the tissue reactions in conventional vaccines, such as whole cellbacterins. The concentration of LPS in the stock antigen of Example 3was found to be negative as examined by the Limulus Amebocyte LysateAssay (SIGMA, Chemical Company, St Louis Mo.).

Example 8 The Effect of Vaccinating Fresh Cows and Cows in FirstLactation

[0128] A subunit vaccine consisting SRPs and porins derived fromSalmonella dublin (strain designation MS010207) and Salmonellatyphimurium (strain designation MS010427) were administered to twogroups of lactating cows in a controlled field study within a largeexpansion dairy. The dairy consisted of 500 cows separated into fivelarge freestall corrals (100 cows/corral) based on days in milk orperiod of lactation. Two groups of cows were chosen for the study; freshcows (30-90 days post-partum) and high-producing heifers (cows in firstlactation). Cows received two subcutaneous vaccinations 28 days apart.The experimental trial examined the safety of the immunizing compositionbased on the tissue reactivity of the injected material at the site ofinjection, the effect vaccination had on milk production, the prevalenceof Salmonella and somatic cell counts between vaccinated andnon-vaccinated cows. Data was collected on performance and physiologicalstatus from individual cows using an integrated electronic cowidentification system.

[0129] Preparation of an Immunizing Composition Derivedfrom Salmonelladublin and Salmonella typhimurium

[0130] The immunizing composition was prepared as described in Example 3with the following modifications. Three high molecular weight SRPs atapproximately the 89 kDa, 84 kDa and 72 kDa and porins in the range of38-39 kDa were harvested from each of the two isolates. The lowermolecular weight IRPs (37 kDa, 32 kDa and 29 kDa) that S. bredeneyexpressed under iron restriction of Example 3 were poorly expressed inS. dublin and/or S. typhimurium and were not present in the final stockantigen as examined on a 10% SDS-Page gel. Nevertheless, the upperbanding profile (89 kDa, 84 kDa and 72 kDa) of these two isolates wereidentical to S. bredeney of Example 3. The immunizing compositionconsisted of equal concentration of SRPs from S. dublin and S.typhimurium so as to provide a total dose of 1000 μg, 500 μg from eachisolate. The antigen solution was emulsified into EMUSIGEN (22.5%vol/vol) as previously described in Example 3.

[0131] Pre-vaccination

[0132] Thirty days before the first vaccination the herd-exposure statusto Salmonella was determined. Fecal samples were collected from eachindividual cow as described in Example 6. The total number of samplescollected was 144 (60 Fresh cows and 84 Heifers). Salmonella wasrecovered from 50% of the Fresh cows and 27% from the cows in firstlactation. Three serotypes were found; S. anatum, S. uganda and S.meleagridis; S. dublin and S. typhimurium were not detected. The SRP andporin profiles of these isolates were found to be identical to thebanding profiles of S. dublin, S. typhimurium and S. bredeney. Becauseof the wide spread incidence of S. dublin and S. typhimurium in thebovine species and the conserved nature of these proteins it was decidedto use these antigens in the vaccine composition to give furtherclarification of the cross-protective nature of these proteins.

[0133] Immunization of Fresh Cows and Cows in First Lactation

[0134] Fifty percent of the cows in first lactation (42 out of 84) and50% of the fresh cows (30 out of 60) were vaccinated. The remaining cowsin each group remained as non-vaccinated controls. Briefly, cows fromeach group were randomly placed in a large holding stanchion. Everyother cow was given a 2 ml intramuscular injection of the vaccine. Inaddition, fecal samples were taken from all cows in each group by rectalextraction at the time of the first vaccination. All suspect isolationswere serotyped as described in Example 6. The somatic cell count andmilk production for each cow was acquired prior to the first vaccinationto establish a historical performance trend. The production of milk fromindividual cows was monitored daily as well as general health andadverse reaction to vaccination. The somatic cell counts were monitoredmonthly by the DHIA. The vaccinated cows were given a second vaccination(Booster) four weeks after the first vaccination. The vaccinated andnon-vaccinated test cows within the herd were identified by ear tags andmilk production was monitored by an electronic cow identification systemusing a transponder, hung on a strap around the cows neck. The overallperformance of the vaccinated and non-vaccinated cows was monitoredthroughout the experimental study.

Results

[0135] The injection sites of vaccinated cows were examined 14 daysafter the first and second vaccination. None of the vaccinated cowsshowed any adverse tissue reaction to the vaccine at the site ofinjection. There was no visible swelling or defined nodule in any of thecows examined. In addition, daily observations of these cows showed novisible changes in behavior and/or activity.

[0136] Fecal samples taken from both groups the day of the firstvaccination revealed a significant decline in the shedding prevalence ofSalmonella as compared to samples taken 30 days before vaccination. Theisolation rate in the fresh cow group declined to 27% while the cows infirst lactation had dropped to 8%. In fact, the isolation rate ofSalmonella at the second vaccination showed no difference betweengroups. Only five isolates of Salmonella were cultured between groups,three from the fresh cow group and 2 from the first lactation cow group.There was no difference in the shedding prevalence of Salmonella betweenthe vaccinated and non-vaccinated cows from either group.

[0137] The yield of milk per cow was monitored daily in both groups.FIG. 6 shows the weekly average milk production between the vaccinatedand non-vaccinated cows in first lactation. There was no statisticaldifference in the yield of milk from the first vaccination (week 1) tothe second vaccination (week 2) when compared to the non-vaccinated cows(P=0.435) and from the second vaccination (week 5) through the 16^(th)week of production (P=0.07) as graphically depicted in FIG. 6. However,in the fresh cow group, the production of milk statistically increasedin the vaccinated cows after each vaccination as compared to thenon-vaccinated group (FIG. 7). The degree of significance from the firstvaccination to the second vaccination was P=0.006 and dramaticallyincreased from the second vaccination to the ₁₆ ^(th) week of production(P=0.000000067). Sixteen weeks after the first vaccination the averagepounds of milk produced per cow in the vaccinated group was 60.3 poundscompared to 56.4 pounds in the non-vaccinated controls. This was a 6.5%increase in milk production over the control group or 3.9 pounds/cowadvantage.

[0138] The somatic cells counts were also positively effected throughvaccination in both the fresh cows and cows in first lactation. FIG. 8shows the monthly average (DHIA) somatic cell counts between thevaccinated and non-vaccinated cows in first lactation, beginning fromthe first vaccination through 16 weeks of production. The data showsthat the vaccinated group had a 30.0% difference in the average somaticcell count with a degree of significance of P=0.036 as compared to thenon-vaccinated control group. This reduction in somatic cell count wasmore dramatically pronounced in the vaccinated fresh cows as illustratedin FIG. 9. The data shows that the level of somatic cells decreased by58.8% (P=0.02) in the vaccinated cows as compared to the non-vaccinatedgroup.

[0139] The difference in performance between the fresh cows and in firstlactation could be due to the difference in the health status of thecow. Typically fresh cows are under a higher degree of stress due totheir physiological status then other cows in production, predisposingthem to a greater disease challenge. Stress can often exasperate thelikelihood of a diseased condition that may effect the overall healthand performance of the animal. It is interesting to note there was nostatistical difference in milk production between the vaccinated andnon-vaccinated cows in first lactation, in contrast to the vaccinatedfresh cows. It would appear that the vaccine had a positive effect onthe health status of the vaccinated fresh cows, as seen by the enhancedmilk production. This enhanced performance did not appear to be relatedto a clinical disease caused by Salmonella since the isolation ratenaturally declined and there was no difference in the prevalence betweenvaccinated and non-vaccinated cows. In addition, the vaccine compositioncontained the immunogens derived from Salmonella dublin and Salmonellatyphimurium and not from the isolates found within the herd. Because ofthe conserved nature of these proteins among gram negative and grampositive bacteria it is highly likely that the vaccine induced a degreeof cross-protection against other bacteria expressing these proteins,allowing the animal to perform better.

Example 9 Immunization of Feed Lot Steers for the Control of Salmonella,Trial-I

[0140] A commercial feed lot having a history of Salmonellosis was usedin a controlled field study to evaluate the efficacy of an immunizingcomposition consisting of SRPs and porins derived from Salmonelladublin. The experimental trial examined the safety of the immunizingcomposition based on the tissue reactivity of the injected material atthe site of injection, the serological response to vaccination, and theshedding prevalence of Salmonella.

[0141] The feed lot consisted of 500 Holstein steers separated intoseparate grow out facilities based on the age and weight of the steers.The experimental trial was initiated in starter calves (N=150) with anaverage weight of approximately 150 pounds. The steers were randomlydistributed into 10 separate pens (1-10) so that each pen contained 15steers. Ear tags individually identified steers in each pen. Theexposure status to Salmonella was determined prior to the firstvaccination. Individual fecal samples were taken from all steers toestablish a shedding prevalence of Salmonella. Samples were processed aspreviously described in Example 6. Salmonella was recovered from 56% ofthe 150 samples taken. Three different serotypes were identified; S.dublin, S. uganda and S. Muenster. Salmonella dublin was the predominantserotype, and was found within the herd at 67%.

[0142] The Salmonella positive steers were identified from each pen anddistributed among four pens (P3, P4, P5, and P6) so that each pencontained the same number of positive and negative steers. Thus, eachpen contained 8 Salmonella positive steers and 7 Salmonella negativesteers so that 53.3% of each pen was Salmonella positive.

[0143] The immunizing compositions was prepared from the SRPs of S.dublin having molecular weights of 89 kDa, 84 kDa, 72 kDa and porinshaving molecular weights of 38-39 kDa. The target proteins wereemulsified into EMULSIGEN (22.5% vol/vol) to provide a total proteindose of a 1000 μg in a 2 ml injectable volume as previously described.

[0144] Steers in pens 3 and 5 received two intramuscular vaccinations 28days apart. Steers in pens 4 and 6 remained as non-vaccinated controls.Blood was taken from 12 steers per pen at day 0 (First vaccination) andagain at 2, 4, and 6 weeks post to monitor the serological response tovaccination. Individual fecal samples were collected from each steer asdescribed in Example 6 on the day of the first vaccination and again at2 and 6 weeks.

[0145] The injection sites of each vaccinated steer were examined 14days after the first and second vaccination. None of the vaccinatedsteers showed any adverse tissue reaction at the site of injection. Inaddition, daily observations of these steers showed no visible changesin behavior and/or activity as compared to the non-vaccinated groups.FIG. 10 shows the serological response of vaccinated steers compared tonon-vaccinated controls as evaluated by ELISA of Example 7. The vaccineinduced elevated antibody titers to SRPs after each vaccination. Therewas a rise in titer after the first vaccination that declined two weeksafter but continued to rise after the second vaccination, clearlydemonstrating that a secondary response was induced.

[0146] Table 5 shows the shedding prevalence of Salmonella betweenvaccinated and non-vaccinated pens. Fecal samples taken from individualsteers on the day of the first vaccination (week 0) revealed asignificant decline in the shedding prevalence of Salmonella in all testgroups as compared to samples taken before vaccination (Table 5). Thedecline in the shedding prevalence continued through the duration of thesampling period in all test groups. However, the shedding prevalence 14days after the last vaccination indicated a difference between thevaccinated group as compared to the non-vaccinated controls. There was ahigher percentage of Salmonella positive steers (29%) in Pen 6 ascompared to the vaccinated pens showing only 6.7%. TABLE 5 The SheddingPrevalence of Salmonella Between Vaccinated and Non-vaccinated Pens Pen3- Pen 4 Pen 5 Pen 6 Sampling time Vaccinated Control Vaccinated ControlPre-vaccination 53.3% 53.3% 53.3% 53.3% 0   33%   40%   47%   43% 2weeks   27%   13%   13%   36% 6 weeks  6.7%   13%  6.7%   29%

Example 10 Immunization of Feed Lot Steers for the Control of SalmonellaTrial-2

[0147] The commercial feed lot of Example 9 was used in a controlledfield study to provide further data on the efficacy of an immunizingcomposition consisting of SRPs and porins derived from Salmonella dublinand Salmonella typhimurium. The experimental trial examined the safetyof the immunizing composition based on the tissue reactivity of theinjected material at the site of injection, the serological response tovaccination, and the shedding prevalence of Salmonella.

[0148] At the end of the experiment of Example 9 the facility wascleaned, sanitized and disinfected and allowed to sit empty for 2 weeksprior to the arrival of a new group of steers. Environmental samples(N=2) were taken from each pen to ascertain the incidence of Salmonella.Samples were cultured as previously described in Example 6. Allenvironmental samples were found negative for Salmonella. One hundredfifty (N=150) 4 month old Holstein steers with and average weight of 300pounds were transported by truck from Idaho. Upon arrival, steers wereunloaded, ear tagged for identification and randomly distributed among10 separate pens (1-10) so that each pen contained 15 steers. One weekafter arrival the exposure status to Salmonella was determined prior tothe first vaccination. Individual fecal samples were taken from allsteers to establish a shedding prevalence of Salmonella. Samples wereprocessed as previously described in Example 6. All of the 150 samplestaken were found negative for Salmonella. Based on this information avaccine composition was prepared from two Salmonella isolates (S. dublin(strain designation MS010207) and S. typhimurium (strain designationMS010427)).

[0149] The immunizing compositions was prepared from the SRPs of S.dublin and S. typhimurium having molecular weights within a range of 89kDa, 84 kDa, 72 kDa and porins having molecular weights within a rangeof 38-39 kDa. The proteins (500 μg from each isolate) were absorbed ontoaluminum hydroxide (25% vol/vol) to provide a total protein dose of a1000 μg in a 2 ml injectable volume.

[0150] As before, steers in pens 3 and 5 received two intramuscularvaccinations 28 days apart. Steers in pens 4 and 6 remained asnon-vaccinated controls. Blood was taken from 12 steers per pen at day 0(First vaccination) and again at 2, 4, and 6 weeks post to monitor theserological response to vaccination. Individual fecal samples werecollected from each steer as described in Example 6 on the day of thefirst vaccination and again at 2 and 6 weeks.

[0151] The injection sites of each vaccinated steer, as before, wereexamined 14 days after the first and second vaccination. None of thevaccinated steers showed any adverse tissue reaction at the site ofinjection using aluminum hydroxide as the adjuvant. In addition, dailyobservations of these steers showed no visible changes in behaviorand/or activity as compared to the non-vaccinated groups. Theserological response to the vaccine was determined as described hereinand compared to the non-vaccinated controls. The vaccine inducedelevated antibody titers to SRPs after each vaccination that wascomparable to the composition of Example 9. There was a rise in titerafter the first vaccination that declined for two weeks after butcontinued to rise after the second vaccination.

[0152] Fecal samples were taken from all steers at the time of firstvaccination and again at 2 and 6 weeks after vaccination. Salmonella wasnot isolated from any of the samples taken during the sampling period.In addition, environmental samples (N=2) taken from each pen at the 6week period were negative for Salmonella.

[0153] Nine weeks after the first vaccination steers in both the controland vaccinated pens were individually weighed. The average weight ofsteers in the control pens were 730.5 lbs (Pen-4) and 745.6 lbs (Pen-6)(Table 6) with an average weight of both pens at 738.0 lbs. In contrast,the average weight of vaccinated steers were 767.7 pounds (Pen-3) and761.8 lbs (Pen-5) (Table 6) with a combined average weight of 764.8 lbs.There was a 26.7 pound advantage in the vaccinated steers as compared tothe steers in the non-vaccinated groups with a degree of significance ofP=0.018. This enhanced weight performance did not appear to be relatedto a clinical disease caused by Salmonella since the organism was notdetected in any of the steers examined. It is believed that theconserved nature of these proteins in the vaccine composition induced adegree of cross-protection against other bacteria expressing theseproteins, thus lessening subclinical diseases, allowing the animal toperform better as seen in the difference in weight between the twogroups. TABLE 6 The Comparison of Individual Weights Between Vaccinatedand Non-Vaccinated steers 9 weeks after the first vaccination Pen-6Pen-3 Vaccinated Pen-4 Control Pen-5 Vaccinated Control Weight Weight inpounds Weight in Pounds Weight in Pounds in Pounds 742 682 816 724 889723 717 812 750 595 716 756 712 705 811 735 844 737 801 779 794 726 740717 769 780 796 744 755 752 758 670 698 811 785 749 772 706 785 775 746778 743 729 744 725 764 819 697 741 719 712 809 744 775 688 795 752 701775 Mean = 767 Mean = 730.5 Mean = 761.8 Mean = 745.6 SD¹ = 52.7 SD =49.7 SD = 37.6 SD = 41.9 CV = 6.9 CV = 6.8 CV = 4.9 CV = 5.7

Example 11 Purification of Siderophore Receptor Proteins ofStaphylococcus Aureus of Human and Avian Origin

[0154] Two field isolates of Staphylococcus aureus and three additionalisolates obtained from the American Type Culture Collection ATCC(isolates 8432, 11371, and 19636) were evaluated for the expression ofsiderophore receptor proteins. Field isolates originating from turkeyswere isolated from the hock joints of diseased birds. ATCC isolate 8432was also of avian origin, while isolates 11371 and 19636 were of humanorigin. All bacteria were grown in Brain heart infusion broth (BHI,Difco) as iron-deplete and/or iron-replete media. The iron-deplete mediawas iron-restricted chemically using 22′-dipyridyl at 175 mM, whereasthe iron-replete media contained 200 μM ferric chloride. The bacteriawere grown in 10 ml of BHI for 8 hours at 37° C. while stirring at 400rpm. At 8 hours of incubation, 1.0 ml of culture was removed and washedin 10 volumes sterile physiological saline by centrifugation (10,000×g)for 10 minutes. The pellet was resuspended in 100 microliters (1 μl) ofsaline containing 1 mg lysostaphin (Sigma, St. Louis, Mo.) was added,and the suspension was then incubated at 37° C. for 2 hours. Thebacterial suspension was centrifuged at 12,000×g for 1 minute. Thesupernatant was collected and centrifuged again at 20,000×g for 40minutes. The bacterial pellet was resuspended in 100 μl tris-bufferedsaline (TBS) at pH 7.4. The resuspended bacterial pellets from thedifferent isolates were resolved by 12% SDS-PAGE and transferred ontonitrocellulose membranes and tested for cross-reactivity with sera toSRPs of gram negative bacteria. Absorbed rabbit polyclonal hyper-immunesera prepared against purified SRPs from E. coli and/or S. typhimuriumwere used as probes in the immunoblot of the S. aureus SRPs.

[0155] The SDS-PAGE patterns of the outer membrane protein extracts ofthe Staphylococcus aureus isolates showed different patterns of SRPexpression between the field isolates and the ATCC isolates. The fieldisolates of turkey origin grown under conditions of iron restrictionshowed four proteins with molecular weights between 66-90 kDa(specifically, 90 kDa, 84 kDa, 72 kDa and 66 kDa) and also at about 36kDa, 32 kDa and 22 kDa regions. The ATCC isolates showed only a singleSRP at the 40-55 kDa range (42 kDa) and at the 36 kDa range. None of theATCC isolates showed an SRP at 66-90 kDa region, including isolate 8432of avian origin.

[0156] Western blot analysis of the isolated SRPs of the S. aureus fieldisolates was conducted by probing with sera raised to the SRPs ofSalmonella (89 kDa, 84 kDa and 72 kDa) and/or E. coli (89 kDa, 84 kDa,78 kDa, and 72 kDa). The sera reacted strongly with the proteins in the66-90 kDa range but not with the lower molecular weight proteins (i.e.,36 kDa, 32 kDa and 22 kDa). The Salmonella sera also reacted with aprotein in the 31 kDa range that appeared to be similar to the 31 kDaprotein of the transmembrane proteins of gram negative bacteria.

[0157] This data indicates that S. aureus expressed SRPs that are withina similar molecular weight range as gram negative bacteria, and thatantibodies raised against SRPs from gram negative bacteria cross-reactbetween at least two different families of bacteria. This composition isused to vaccinate animals as described herein, and the ability of thecomposition to protect animals from homologous and heterologouschallenge is determined, as well as the ability of the composition toenhance performance characteristics of the animal.

[0158] The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

[0159] All headings are for the convenience of the reader and should notbe used to limit the meaning of the text that follows the heading,unless so specified.

1-46. (Canceled)
 47. A method for treating mastitis in a milk producinganimal, the method comprising administering to a milk producing animalhaving or at risk of having mastitis an effective amount of acomposition comprising: at least two SRPs isolated from a gram negativemicrobe; at least two porins isolated from the gram negative microbe;Lps at a concentration of no greater than about 10.0 EU/ml; and apharmaceutically acceptable carrier:
 48. The method of claim 47 whereinthe gram negative microbe is an enteropathogen.
 49. The method of claim47 wherein the gram negative microbe is a member of the familyEnterobacteriaceae.
 50. The method of claim 47 wherein the gram negativemicrobe is a member of the tribe Escherichieae or Salmonelleae.
 51. Themethod of claim 47 wherein the gram negative microbe is Salmonella spp.or Escherichia coli.
 52. The method of claim 47 wherein the at least twoSRPs have molecular weights of between about 60 kDa and about 100 kDa.53. The method of claim 47 wherein the at least two porins havemolecular weights of between about 30 kDa and about 43 kDa
 54. Themethod of claim 47 wherein the animal is a bovine. 55-71. (Canceled)